Compositions and methods for the treatment of viral infections

ABSTRACT

The invention provides compositions, kits and methods utilizing polypeptides having a viral alpha-helix heptad repeat domain in a stabilized α-helical structure (herein also referred to as SAH). The compositions are useful for treating and/or preventing viral infections. The invention is based, at least in part, on the result provided herein demonstrating that viral hydrocarbon stapled alpha helical peptides display excellent proteolytic, acid, and thermal stability, restore the native alpha-helical structure of the peptide, are highly effective in interfering with the viral fusogenic process, and possess superior pharmacokinetic properties compared to the corresponding unmodified peptides.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2009/000438 (WO2009/108261) filed on Jan. 23, 2009 which claimspriority to U.S. Provisional Patent Application Ser. No. 61/062,007filed on Jan. 23, 2008 which is incorporated herein by reference in itsentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web, and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 24, 2011, isnamed 69208US7.txt and is 79,397 bytes in size.

BACKGROUND

The molecular process of viral fusion, in which viral coat proteinsrecognize and bind to surface receptors of the host cell, is a criticaltarget in the prevention and treatment of viral infections. Uponrecognition of the viral glycoprotein by host cellular receptors, viralfusion proteins undergo conformational changes that are essential toviral fusion and infection. A series of hydrophobic amino acids, locatedat the N- and C-termini organize to form a complex that pierces the hostcell membrane. Adjacent viral glycoproteins containing two amphipathicheptad repeat domains fold back on each other to form a trimer ofhairpins, consisting of a bundle of six α-helices. This six-helix bundlemotif is highly conserved among many viral families, including Filovirus(ebola), (Malashkevich, V. N., et al., PNAS, 1999. 96(6): p. 2662-2667;Weissenhorn, W., et al., Molecular Cell, 1998. 2(5): p. 605-616),Orthomyxovirus (influenza) (Wilson, I. A., J. J. Skehel, and D. C.Wiley, Nature, 1981. 289(5796): p. 366-37; Bullough, P. A., et al.,Nature, 1994. 371(6492): p. 37-43), Coronavirus (SARS) (Xu, Y. H., etal. Journal of Biological Chemistry, 2004. 279(47): p. 49414-49419),Paramyxovirus (HRSV) (Zhao, X., et al., PNAS, 2000. 97(26): p.14172-14177) and Retrovirus (HIV) (Chan, D. C., et al., Cell, 1997.89(2): p. 263-27; Weissenhorn, W., et al., Nature, 1997. 387(6631): p.426-430).

HIV envelope proteins gp120 and gp41 non-covalently associate with eachother to form a trimer of dimers. On the host cell, gp120 specificallyinteracts with CD4, CXCR4, and CCR5, which are the glycoproteinsinvolved in host-cell recognition. gp41, the viral membrane spanningglycoprotein, is responsible for fusing the viral and cellularmembranes, resulting in viral particle uptake by the host cell. Oncegp120 binds to CD4, gp41 undergoes a conformational change, transformingfrom its native state into a fusogenic six-helix bundle. The regions ofgp41 involved in this change are 43 (C43) residues of the C-terminalheptad repeat (CHR or HR-2), near the transmembrane domain, and 51 (N51)residues of the N-terminal heptad repeat (NHR or HR-1), found justproximal to the fusion peptide domain. Peptides N51 and C43 orient toform helical antiparallel heterodimers, which associate to form a higherorder trimeric complex that is thermo- and proteolytically stable.

Peptides which interfere with this viral fusogenic process can be usedfor the prevention and treatment of viral infections. For example,peptides corresponding to residues 553-590 of the gp41 N-terminal heptadrepeat domain (HR-1) and residues 630-659 and 648-673 of the C-terminalheptad repeat domain (HR-2) of HIV have been shown to inhibit thereplication of a variety of HIV strains. Studies have determined thatthese peptides inhibit cell-cell fusion by interacting with the HIVenvelope glycoproteins.

T20 or enfuvirtide, is the first fusion inhibitor peptide developedbased on the CHR region of gp41 for the treatment of HIV. Enfuvirtide isactive at nanomolar concentrations against many strains and subtypes ofHIV, including the common lab strains and primary isolates of HIV-1 andHIV-2 (Wild, C. T., et al., PNAS, 1994. 91(21): p. 9770-9774).

However, enfuvirtide has remained a tertiary treatment option due to avariety of factors which include cost, no oral bioavailability(subcutaneous injections limit accessibility and compliance) and poor invivo stability (Kilby, J. M., et al., Nuclic Aids Research and HumanRetroviruses, 2002. 18(10): p. 685-693), and loss of bioactive secondarystructure. Thus, although peptide-based inhibition of viral fusionprocesses is mechanistically feasible and clinically effective, thebiophysical and biochemical properties of amphipathic fusion peptidespresent numerous challenges which hinder their use.

SUMMARY OF THE INVENTION

The present invention is directed to compositions, kits and methodsutilizing polypeptides with stabilized α-helical structures (herein alsoreferred to as SAH). The compositions are useful for treating and/orpreventing viral infections. The invention is based, at least in part,on the result provided herein demonstrating that viral hydrocarbonstapled alpha helical peptides display excellent proteolytic, acid, andthermal stability, restore the native alpha-helical structure of thepeptide, are highly effective in interfering with the viral fusogenicprocess, and possess superior pharmacokinetic properties compared to thecorresponding unmodified peptides.

In a first aspect, the invention is directed to a modified polypeptidehaving a stabilized viral alpha helix heptad repeat domain. Preferablythe alpha helix heptad repeat domain is stabilized with at least onehydrocarbon staple, but could include two, three or more hydrocarbonstaples. Suitable hydrocarbon staples (e.g., tethers) are describedherein. Suitable viral alpha helix heptad repeat domains are derivedfrom any virus with an alpha helix domain or analog thereof that isdirectly or indirectly involved in cell attachment and/or fusion.Suitable stabilized alpha helical heptad repeat domains can be derivedfrom numerous viruses, including respiratory syncytial virus,parainfluenza virus, influenza virus, coronavirus, ebolavirus and HIV.The modified polypeptides of the invention can include a stabilized HIVgp41 heptad repeat domain (e.g., heptad repeat domain 1 or 2, orportions thereof).

Any of the modified polypeptides of the invention can be included incompositions and kits.

In another aspect, the invention is directed to a method for inhibitingthe transmission of HIV to a cell. In the method, the HIV virus iscontacted with an effective dose of a modified polypeptide so that theHIV virus is inhibited from infecting the cell. Preferably, the modifiedpolypeptide has an HIV gp41 heptad repeat domain (e.g., heptad repeatdomain 1 or 2, or portions thereof) that is stabilized with ahydrocarbon staple.

The invention may also include a method for treating or delaying theonset of AIDS in an HIV infected individual. A pharmaceuticalcomposition having a modified polypeptide with a stabilized HIV gp41heptad repeat domain (e.g., heptad repeat domain 1 or 2, or portionsthereof) is administered to an individual infected with HIV, thustreating or delaying the onset of AIDS. Preferably the HIV gp41 heptadrepeat domain is stabilized with a hydrocarbon staple.

In still another aspect, the invention is directed to a method forincreasing the number of CD4+ cells in an individual infected with HIV.The method involves administering to the individual infected with HIV aneffective dose of a pharmaceutical composition having a modifiedpolypeptide with a stabilized HIV gp41 heptad repeat domain (e.g.,heptad repeat domain 1 or 2, or portions thereof). The administration ofthe composition results in an increase in the number of CD4+ cells inthe individual. Preferably the HIV gp41 heptad repeat domain isstabilized with a hydrocarbon staple.

In yet another aspect, the invention is directed to a method forinhibiting syncytia formation between an HIV infected cell and anuninfected cell. The method involves contacting the infected cell withan effective dose of a modified polypeptide having a stabilized HIV gp41heptad repeat domain (e.g., heptad repeat domain 1 or 2, or portionsthereof), thereby inhibiting syncytia formation between the cells.Preferably the HIV gp41 heptad repeat domain is stabilized with ahydrocarbon staple.

In still another aspect, the invention is directed to a method forinactivating HIV. The method involves contacting the virus with aneffective dose of a modified polypeptide having a stabilized HIV gp41heptad repeat domain (e.g., heptad repeat domain 1 or 2, or portionsthereof) so that the HIV is rendered inactive (e.g., non-infectious).Preferably the HIV gp41 heptad repeat domain is stabilized with ahydrocarbon staple.

In still another aspect, the invention is directed to a method forpreventing an HIV infection in an individual. The method involvesadministering to an individual an effective dose of a pharmaceuticalcomposition having modified polypeptide with a stabilized HIV gp41heptad repeat domain (e.g., heptad repeat domain 1 or 2, or portionsthereof). Administration of the stabilized HIV gp41 heptad repeat domaininterferes with the ability of the HIV to infect the individual.Preferably the HIV gp41 heptad repeat domain is stabilized with ahydrocarbon staple.

The modified polypeptides can be used to inhibit the transmission of RSVto a cell. The virus is contacted with an effective dose of a modifiedpolypeptide having a stabilized RSV viral alpha helix heptad repeatdomain analog thereby inhibiting transmission of the virus to a cell.Preferably the heptad repeat domain analog is stabilized with thehydrocarbon staple.

The modified polypeptides can also be used to inhibit the transmissionof a parainfluenza virus to a cell. The virus is contacted with aneffective dose of a modified polypeptide having a stabilizedparainfluenza viral alpha helix heptad repeat domain analog, therebyinhibiting transmission of the virus to a cell. Preferably the heptadrepeat domain analog is stabilized with the hydrocarbon staple.

In another aspect, the modified polypeptides can also be used to inhibitthe transmission of an influenza virus to a cell. The virus is contactedwith an effective dose of a modified polypeptide having a stabilizedinfluenza viral alpha helix heptad repeat domain analog, therebyinhibiting transmission of the virus to a cell. Preferably the heptadrepeat domain analog is stabilized with the hydrocarbon staple.

In still another aspect, the invention is directed to a method forinhibiting the transmission of a coronavirus to a cell. The methodincludes contacting the coronavirus with an effective dose of a modifiedpolypeptide having a stabilized coronavirus alpha helix heptad repeatdomain analog, thereby inhibiting transmission of the virus to a cell.Preferably the heptad repeat domain analog is stabilized with thehydrocarbon staple.

In yet still another aspect, the invention is directed to a method forinhibiting the transmission of an ebolavirus to a cell. The methodincludes contacting the ebolavirus with an effective dose of a modifiedpolypeptide having a stabilized ebolavirus alpha helix heptad repeatdomain analog, thereby inhibiting transmission of the virus to the cell.Preferably the heptad repeat domain analog is stabilized with ahydrocarbon staple.

In an aspect of the invention, the invention provides modified peptidesof the inventions as a pharmaceutical composition. In some embodiments,the pharmaceutical composition is for enteral administration, preferablyoral administration.

In yet another aspect, the alpha helix heptad repeat domains and analogsthereof are used to generate an antibody response to the polypeptides byadministering the polypeptides to a subject. Furthermore, the antibodiesgenerated directly or indirectly (e.g., humanized antibodies) by theadministration of the polypeptides may then be used to prevent or treata viral infection (e.g., HIV, RSV, parainfluenza, influenza,coronavirus, ebolavirus).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the domains of the gp41 glycoprotein.

FIGS. 2A and B illustrate the amino acid sequence for A) HX-strain ofgp160 (SEQ ID NO: 49) and B) YU2-strain of gp160 (SEQ ID NO: 50), withHR-1 domain bolded and underlined and HR-2 domain bolded and italicized.

FIG. 3 illustrates the amino acid sequences for HIV-1 gp41 HR-1 and HR-2domains and homologous regions in other viruses (SEQ ID NOS: 14, 7, 9,11, 51-54, 8, 10, 12 and 55-57, respectively, in order of appearance).

FIG. 4A illustrates the HIV six-helix bundle and key interhelixinteractions of the helicies N36 and C34. One of the N36 and two C34helicies are faded for clarity. The helical wheel further illustrateskey contacts among the helicies based upon the a, b, c, d, e, f, g,nomenclature.

FIG. 4B illustrates the fusogenic bundle formed by HR-analog domainsfrom RSV, influenza, SARS and Ebola. The six-helix fusogenic bundle ishighly conserved across many species.

FIG. 5A provides examples of amino acid sequence templates from withinthe HIV-1 HR-2 domain polypeptides with sequential N-terminaltruncations. Shown are N-terminal truncations of the polypeptides of SEQID NOS: 1, 13, 58, and 59, respectively.

FIG. 5B provides examples of amino acid sequence templates from withinthe HIV-1 HR-2 domain polypeptides with sequential C-terminaltruncations. Shown are C-terminal truncations of the polypeptides of SEQID NOS: 1, 13, 58, and 59, respectively.

FIG. 6 provides examples of sequence templates from within the HIV-1 HR2domain depicting staggered N- and C-terminal truncations. Shown arestaggered N- and C-terminal truncations of the polypeptides of SEQ IDNOS: 1, 13, 58, and 59, respectively.

FIG. 7 illustrates a synthetic design of a truncated SAH-gp41 compound,SAH-gp41₍₆₂₆₋₆₄₅₎ (A) SEQ ID NO: 61 which is based on gp41₍₆₂₆₋₆₄₅₎ SEQID NO: 60 X=S5 amino acid, B=norleucine.

FIG. 8 provides examples of sequence templates from within the HR2domains of SIV (SEQ ID NO: 62) and the HX (SEQ ID NOS: 1 and 13) and YU2(SEQ ID NOS: 58 and 59) strains of HIV-1 depicting the generation ofchimeras (SEQ ID NO: 62).

FIG. 9 illustrates the heptad repeat domain motif as applied to HIV gp41(626-663) (SEQ ID NO:66) and associated preferred amino acid residues.Examples of sequence template from within the HIV-1 HR2 domain depictingthe specific amino acid residues necessary to preserve the HR1interaction are provided (SEQ ID NOS: 44 and 45). Thus, the positionsindicated with a dash may be amenable to substitution/mutation withoutdisruption of activity.

FIGS. 10A-D illustrate the possible combinations of helix-stabilizingcrosslinks formed at positions A) i, and i+4 across one turn in thehelix using two S5 amino acids; B) i, and i+7, across two turns of thehelix using one S8 and one R5 amino acid or one R8 and one S5 aminoacid; C) a double crosslink employing two i, i+4, two i, i+7, or one i,i+4 and one i, i+7 crosslink; and D) a triple crosslink employing anycombination of i, i+4, i, i+7, or other crosslinks (e.g. i, i+3).

FIG. 11 illustrates SAH-gp41 singly stapled peptides (SEQ ID NOS: 67-71,1, and 72-79, in order of appearance). (e.g., N-term: Ac, FITC-βAla,Biotin-τAla; C-term: CONH₂, COOH). X=S5 amino acid, B=norleucine

FIG. 12 includes sequences of doubly and triply stapled SAH gp41peptides (SEQ ID NOS 80-87, 1 and 88-98, in order of appearance). (e.g.,N-term: Ac, FITC-βAla, Biotin-βAla; C-term: CONH₂, COOH). X=S5 aminoacid, B=norleucine.

FIG. 13 illustrates unstapled, singly stapled and doubly stapled gp41HR-2 peptides (SEQ ID NOS 99-100 and 82-85, in order of appearance) andillustrates a strategy for locating the staples in the helix. Staplesare positioned so as to preserve and/or optimize inter-helix interactionsurfaces. X=S5 amino acid, B=norleucine

FIGS. 14A-F illustrate that singly and doubly stapled SAH-gp41 compoundsexhibit greater helical stability as compared to the unmodified gp41peptides at pH 7 and pH2. Percent helicity for each compound isindicated in parenthesis; A) SAH-gp41₍₆₂₆₋₆₆₂₎ singly- anddoubly-stapled peptides at pH 7, B) SAH-gp41₍₆₃₈₋₆₇₃₎ singly-stapledpeptides at pH 7, C) SAH-gp41₍₆₃₈₋₆₇₃₎ doubly-stapled peptides at pH 7,D) SAH-gp41₍₆₂₆₋₆₆₂₎ singly- and doubly-stapled peptides at pH 2, E)SAH-gp41₍₆₃₈₋₆₇₃₎ singly- and doubly-stapled peptides at pH 2, F) Tablecomparing calculated percent helicities of SAH-gp41 compounds at pH 7and pH 2.

FIGS. 15A-C illustrate that singly and doubly stapled SAH-gp-41compounds exhibit greater thermal stability compared to the unmodifiedgp41 peptides at pH 7 A) select singly- and doubly-stapledSAH-gp41₍₆₂₆₋₆₆₂₎ compounds; B) singly-stapled SAH-gp41₍₆₃₈₋₆₇₃₎compounds; and C) doubly-stapled SAH-gp41₍₆₃₈₋₆₇₃₎.

FIGS. 16A-F illustrate that SAH-gp41 compounds exhibit greater proteaseresistance to chymotrypsin at pH 7 and pepsin at pH 2 compared to theunmodified gp41 peptides; A) SAH-gp41₍₆₂₆₋₆₆₂₎, chymotrypsin pH 7, B)SAH-gp41₍₆₃₈₋₆₇₃₎ chymotrypsin pH 7, C) Table of half-lives of SAH-gp41compounds in the presence of chymotrypsin, pH 7, D) SAH-gp41₍₆₂₆₋₆₆₂₎,pepsin pH 2, E) SAH-gp41₍₆₃₈₋₆₇₃₎ pepsin, pH 2, F) Table of half-livesof SAH-gp41 compounds in the presence of pepsin, pH 2

FIG. 17 shows a fluorescence polarization binding analysis of HIV fusioninhibitor peptides to the gp41 five-helix bundle illustrating enhancedbinding of SAH-gp41 to the five-helix bundle compared to the unmodifiedpeptides.

FIG. 18 shows improved inhibition of syncytia formation by a truncatedSAH-gp41 compound (A) compared to enfuvirtide (T20: gp41₍₆₃₈₋₆₇₃₎₎,highlighting the potential to retain, and even enhance, anti viralactivity with shorter, stapled peptides.

FIG. 19 demonstrates the anti-viral activity of select SAH-gp41compounds against HIV strains HXBc2, ADA, and HXBc2P 3.2, and YU2. AMLVserves as a negative control.

FIGS. 20A-B demonstrate that A) SAH-gp41 compounds overcome HIV-1 HR1resistance mutations that block the binding of unmodified gp41-basedfusion peptides. Tabulated values indicate fraction of HR2 peptide inputbound to the indicated FITC-HR1 peptide; and B) Select SAH-gp41compounds are notably superior to the corresponding unmodified peptidesin blocking the infectivity of a resistant HIV-1 strain, YU2.

FIG. 21 shows that a doubly-stapled gp41 peptide has markedly enhancedpharmacologic properties in vivo (stability and bioavailability)compared to the corresponding unmodified peptide.

DETAILED DESCRIPTION

The present invention is directed to compositions, kits and methodsutilizing polypeptides with stabilized alpha helical structures. Thecompositions are useful for treating and/or preventing viral infections.The invention is based, at least in part, on the results provided hereindemonstrating that viral hydrocarbon stapled alpha helical peptides haveexcellent structural, proteolytic, acid, and thermal stability, arehighly effective in interfering with virus/cell fusion, and havesuperior pharmacologic properties in vivo compared to their unmodifiedcounterparts.

The alpha helix heptad repeat domain is stabilized with at least onehydrocarbon staple, but could include two, three or more hydrocarbonstaples. The inclusion of multiple hydrocarbon staples is particularlysuited for alpha helical peptides that are 20 or more amino acids inlength. In fact the inclusion of two more hydrocarbon staples, as shownherein, provides for exceptional structural, acid and thermal stabilityof the modified polypeptides, yielding bioactive peptides withstrikingly enhanced pharmacologic properties in vivo.

DEFINITIONS

As used herein, the term “hydrocarbon stapling”, refers to a process forstably cross-linking a polypeptide having at least two modified aminoacids that helps to conformationally bestow the native secondarystructure of that polypeptide. Hydrocarbon stapling allows apolypeptide, predisposed to have an alpha-helical secondary structure,to maintain its native alpha-helical conformation. This secondarystructure increases resistance of the polypeptide to proteolyticcleavage and heat, and also may increase hydrophobicity. Accordingly,the hydrocarbon stapled (cross-linked) polypeptides described hereinhave improved biological activity relative to a correspondingnon-hydrocarbon stapled (uncrosslinked) polypeptide. For example thecross-linked polypeptide can include an alpha-helical domain of an HIVpolypeptide (e.g., HR-1/HR-2 domain), which can interfere with HIVattachment, fusion with, and infection of a cell. In some instances, thecross-linked polypeptide can be used to inhibit virus entry into a cell.The cross-linked polypeptides described herein can be usedtherapeutically, e.g., to treat HIV infection and/or AIDS.

The hydrocarbon stapled polypeptides include one or more tethers(linkages) between two non-natural amino acids, which tethersignificantly enhances the alpha helical secondary structure of thepolypeptide. Generally, the tether extends across the length of one ortwo helical turns (i.e., about 3.4 or about 7 amino acids). Accordingly,amino acids positioned at i and i+3; i and i+4; or i and i+7 are idealcandidates for chemical modification and cross-linking. Thus, forexample, where a peptide has the sequence . . . X1, X2, X3, X4, X5, X6,X7, X8, X9 . . . , cross-links between X1 and X4, or between X1 and X5,or between X1 and X8 are useful as are cross-links between X2 and X5, orbetween X2 and X6, or between X2 and X9, etc. The use of multiplecross-links (e.g., 2, 3, 4 or more) is also contemplated. The use ofmultiple cross-links is very effective at stabilizing and optimizing thepeptide, especially with increasing peptide length, as is the case forsome gp41 fusion peptides. Thus, the invention encompasses theincorporation of more than one crosslink within the polypeptide sequenceto either further stabilize the sequence or facilitate the structuralstabilization, proteolytic resistance, acid stability, thermalstability, and biological activity enhancement of longer polypeptidestretches.

The term “stable” or “stabilized”, as used herein with reference to apolypeptide, refers to polypeptides which have been hydrocarbon-stapledto maintain their natural alpha-helical structure and/or improveprotease resistance and/or improve acid stability and/or improve thermalstability.

As used herein, “HIV” is meant to include HIV-1 and HIV-2 and SIV.“HIV-1” means the human immunodeficiency virus type-1. HIV-1 includesbut is not limited to extracellular virus particles and the forms ofHIV-1 associated with HIV-1 infected cells. “HIV-2” means the humanimmunodeficiency virus type-2. HIV-2 includes but is not limited toextracellular virus particles and the forms of HIV-2 associated withHIV-2 infected cells. The term “SIV” refers to simian immunodeficiencyvirus which is an HIV-like virus that infects monkeys, chimpanzees, andother nonhuman primates. SIV includes but is not limited toextracellular virus particles and the forms of SIV associated with SIVinfected cells.

As used herein a “heptad repeat domain” and “HR domain” refers to apolypeptide that forms an alpha-helix when properly folded. The terms,“heptad repeat domain” and “HR domain” include “HR-like” and “HR-analog”polypeptides. Numerous viral proteins involved in cell attachment andfusion contain HR, HR-like and HR-analog domains including, HIV,parainfluenza, coronavirus, and others. Generally, HR domains arederived from gp41 of HIV, while HR-analog domains are derived from theenvelope glycoproteins of non-HIV viruses. Many HR and HR-analog domainpolypeptides are known in the art and described herein. In oneembodiment, the HR domain has an amino acid sequence which is 40%, 50%,60%, 70%, 80%, or more identical to FIG. 5, FIG. 6 or SEQ ID NO:1-14. Itshould be noted that HR and HR-like domains may have low homology butwill share a common alpha helical structure, with more conservation onthe interaction surfaces than non-interacting surfaces (see FIGS. 4 and9).

In one embodiment, the HR modified polypeptide includes a heptad repeatdomain having the formula: a b c d e f g, wherein a and d arehydrophobic amino acid residues and b, c, e, f and g are any amino acid.Preferably, the formula is repeated in tandem two or more times.

For example, in a further embodiment the heptad repeat domain of themodified polypeptide has the formula: W(a), b, c, W(d), e, f, g, I(a),b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f, g, S(a), b, c, Q(d), e, f,g, N(a), b, c, E(d), e, f, g, L(a), or conservative amino acidsubstitutions thereof and wherein the b, c, e, f and g can be any aminoacid (e.g., residues 3-38 of SEQ ID NO: 44).

In a further, embodiment the heptad repeat domain of the modifiedpolypeptide has the formula: T(g), W(a), b, c, W(d), D(e), R(f), g,I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g, a, Q(b), c, d,Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g), L(a), or conservativeamino acid substitutions thereof and wherein non-designated amino acidscan be any amino acid (e.g., residues 2-38 of SEQ ID NO: 45).

The HR regions are known to comprise a plurality of 7 amino acid residuestretches or “heptads” (the 7 amino acids in each heptad designated “a”through “g”), wherein the amino acids in the “a” position and “d”position are generally hydrophobic. Generally the HR region will includeone or more leucine zipper-like motifs (also referred to as “leucinezipper-like repeats”) comprising an 8 amino acid sequence initiatingwith, and ending with, an isoleucine or leucine. Heptads and leucinezipper like-motifs contribute to formation of a coiled coil structure ofgp41, and of a coiled coil structure of peptides derived from the HRregions. Generally, coiled coils are known to be comprised of two ormore helices that wrap around each other in forming oligomers, with thehallmark of coiled coils being a heptad repeat of amino acids with apredominance of hydrophobic residues at the first (“a”) and fourth (“d”)positions, charged residues frequently at the fifth (“e”) and seventh(“g”) positions, and with the amino acids in the “a” position and “d”position being primary determinants that influence the oligomeric stateand strand orientation (see, e.g., Akey et al., 2001, Biochemistry,40:6352-60).

The effect on stability and oligomerization state of a model coiledcoil, by substituting various amino acids at various positions includingthe “a” and “d” positions, have been reported previously, whereinformation of a trimeric structure was particularly dependent on thesubstitution at the “d” position (see, e.g., Tripet et al., J. Mol.Biol. 300:377-402 (2000); Wagschal et al., J. Mol. Biol. 285:785-803(2000); and Dwyer et al., PNAS USA. 104; 12772-12777 (2007).

It will be apparent to one skilled in the art that any peptide derivedfrom the native sequence of the HR1 domain or HR2 domain of HIV gp41which has antiviral activity (as can be determined using methodsstandard in the art without undue experimentation), and which containsall or a fraction of the region can be used as a native sequence intowhich one or more amino acid substitutions, preferably conservative, inthe domain may be introduced to produce a synthetic peptide providedwith the present invention. For purposes of illustration, such HR2peptides derived from the native sequence, and from which a syntheticpeptide may be produced, may include, but are not limited to, thoseillustrated in FIGS. 5 and 6.

It is apparent to those of ordinary skill in the art that some HR andHR-analog domain residues are less prone to substitution while othersare more accepting of changes. For example, it is preferable not tomutate or to only conservatively mutate the amino acids at positions aand d of the heptad repeat (See FIG. 9). In one embodiment, the heptadrepeat domain has the formula a, b, c, d, e, f, g, wherein a and d arehydrophobic amino acids. In a further embodiment, the heptad repeatdomain has two or more repeats of the formula a, b, c, d, e, f, g. Forexample, in one embodiment the HR domain will have the amino acidsequences illustrated in FIG. 9 or conservative substitutions thereof.Thus, the HR and HR-like domains have significant variability in aminoacid sequence but will maintain an alpha helical structure and antiviralactivity.

In one embodiment, the modified polypeptide includes a heptad repeatdomain having the formula: a b c d e f g, wherein a and d arehydrophobic amino acid residues and b, c, e, f and g are any amino acid.Preferably, the formula is repeated in tandem two or more times.

For example, in a further embodiment the heptad repeat domain of themodified polypeptide has the formula: W(a), b, c, W(d), e, f, g, I(a),b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f, g, S(a), b, c, Q(d), e, f,g, N(a), b, c, E(d), e, f, g, L(a), or conservative amino acidsubstitutions thereof and wherein the b, c, e, f and g can be any aminoacid (e.g., residues 3-38 of SEQ ID NO: 44).

In a further, embodiment the heptad repeat domain of the modifiedpolypeptide has the formula: T(g), W(a), b, c, W(d), D(e), R(f), g,I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g, a, Q(b), c, d,Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g), L(a), or conservativeamino acid substitutions thereof and wherein non-designated amino acidscan be any amino acid (e.g., residues 2-38 of SEQ ID NO: 45).

The HR, HR-like and HR-analog domains are readily identifiable by thosepossessing ordinary skill in the art by sequence based homology,structural homology and/or functional homology. Such methods are wellknown in the art and include bioinformatics programs based on pairwiseresidue correlations (e.g., on the world wide web at:ch.embnet.org/software/COILS_form.html), which have the ability torecognize coiled coils from protein sequences and model their structures(See Lupas, A., et al. Science 1991. 252(5009); p. 1162-1164).Additional methods for identifying HR, HR-like and HR-analog domains aredescribed in U.S. Pat. No. 6,824,783; U.S. Pat. No. 7,273,614; U.S. Pat.No. 5,464,933; and U.S. Pat. No. 7,122,190, all of which are hereinincorporated by reference in their entirety.

In one embodiment, the modified polypeptide of the invention is 70% ormore similar at the interacting face to the amino acid sequence of SEQID NO:1-14, FIG. 5 or FIG. 6. The “interacting face” of the alpha helixincludes those amino acid residues which interact with other amino acidresidues. For example, in the HIV gp41 HR-2 domain the interacting faceincludes the “a” and “d” position amino acids (See FIGS. 4A and 9),while the interacting face of the HIV gp41 HR-1 domain includes aminoacids at positions e, g that interact with HR-2 and a, d that engage inHR1-HR1 interactions (See FIG. 4A). Methods for identifying heptadrepeats and the interacting face residues are well known in the art anddescribed herein.

An “HR-1 domain of HIV” or “heptad repeat one domain of HIV” is anN-terminal portion of the gp41 protein of HIV (the transmembrane subunitof HIV envelope) that forms an alpha-helix when properly folded. TheHR-1 domain of HIV gp41 can include between 5 and 55 amino acid residuesand is based on the sequence of the native HR-1 domain of HIV gp41, or acombination or chimera thereof. The HR-1 domain of HIV can include theN36 domain which encompasses amino acid residues 546-581 HIV-1 Env (SeeFIG. 2 and Bewley et al., J. Biol. Chem. 277:14238-14245 (2002)). HR-1domain polypeptides are known in the art and described herein. In oneembodiment, the HR-1 domain has an amino acid sequence which is 30% ormore identical to SEQ ID NO:2 or 14.

An “HR-2 domain of HIV” or a heptad repeat two domain of HIV is aC-terminal portion of the gp41 protein of HIV (the transmembrane subunitof HIV envelope) that forms an alpha-helix when properly folded. TheHR-2 domain of HIV can include the C34 domain which encompasses aminoacid residues 628-661 of HIV-1 Env (See FIG. 2). HR-2 domainpolypeptides are known in the art and described herein. In oneembodiment, the HR-2 domain has an amino acid sequence which is 40% ormore identical to SEQ ID NO:1 or 13.

As used herein, the term “chimera” or “chimeric”, with reference to thepolypeptides of the invention refers to a polypeptide having at leasttwo different HR domains or having a single HR domain region that iscombined in a manner not found in nature (FIG. 8). For example, thechimera polypeptide may have a first portion of an HIV-1 gp41 HR-2domain and a second portion from a SIV gp41 HR-2 domain. These chimericpolypeptides are encoded by nucleotide sequences which can be been fusedor ligated together resulting in a coding sequence which does not occurnaturally. The chimera includes any functional derivative, fragments,variants, analogues, or chemical derivatives which may be substantiallysimilar to the wild-type HR polypeptides (HIV-1 gp41 HR-2) and whichpossess similar activity (i.e., most preferably, 90%, more preferably,70%, preferably 40%, or at least 10% of the wild-type HR activity, e.g.,inhibiting fusion, viral infectivity).

The terms “treat,” and “treating,” as used herein, shall mean decrease,suppress, attenuate, diminish, arrest, or stabilize the development orprogression of a disease or decrease the occurrence of pathologicalcells (e.g., infected cells) in an animal who is infected with the viraldisorder. The treatment may be complete, e.g., the total absence of HIVin a subject. The treatment may also be partial, such that theoccurrence of infected cells in a subject is less than that which wouldhave occurred without the present invention. Treatment results in thestabilization, reduction or elimination of the infected cells, anincrease in the survival of the patient or decrease of at least one signor symptoms of the disease.

The terms “prevent,” “preventing,” and “prevention,” as used herein,shall refer to a decrease in the occurrence of a disease, or decrease inthe risk of acquiring a disease, or a decrease in the presentation of atleast one sign or associated symptom of the disease in a subject. Theprevention may be complete, e.g., the total absence of disease orpathological cells in a subject. The prevention may also be partial,such that the occurrence of the disease or pathological cells in asubject is less than that which would have occurred without the presentinvention.

The term “inhibits” as used herein with reference to a viral infectionrefers to a decrease in viral transmission, decrease in virus binding toa cellular target or decrease in disease. For example, the polypeptidesof the present invention are used to inhibit viral transmission,syncytia formation, and disease associated with the virus (e.g. AIDS). Acompound of the invention can be screened by many assays, known in theart and described herein, to determine whether the compound inhibits thevirus (e.g., infectivity, transmission, etc.). For example, a compoundof the invention can be assayed for its ability to inhibit viralinfectivity by contacting a cell culture that is incubated with thevirus with a test compound. The compound is found to inhibit viralinfectivity when viral infectivity is 90%, 80%, 75%, 70%, 60%, 50%, 40%,30%, 20%, 10%, 5% or less in the presence of the test compound ascompared to a suitable control (population of cells not subjected toinhibitor).

The term “inhibit transmission”, as used herein, refers to the agent'sability to inhibit viral infection of cells, via, for example, cell-cellfusion or free virus infection. Such infection may involve membranefusion, as occurs in the case of enveloped viruses, or some other fusionevent involving a viral structure and a cellular structure.

The term “inhibiting syncytia formation”, as used herein, refers to anagent's ability to inhibit or reduce the level of membrane fusion eventsbetween two or more moieties relative to the level of membrane fusionwhich occurs between said moieties in the absence of the agent. Themoieties may be, for example, cell membranes or viral structures, suchas viral envelopes.

The terms “effective amount,” or “effective dose” refers to that amountof an agent to produce the intended pharmacological, therapeutic orpreventive result. The pharmacologically effective amount results in theamelioration of one or more symptoms of a viral disorder, or preventsthe advancement of a viral disease, or causes the regression of thedisease or decreases viral transmission. For example, a therapeuticallyeffective amount preferably refers to the amount of a therapeutic agentthat decreases the rate of transmission, decreases HIV viral load, ordecreases the number of infected cells, by at least 5%, preferably atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 35%, at least 40%, at least 45%, at least 50%, at least 55%, atleast 60%, at least 65%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, or more. A therapeuticallyeffective amount, with reference to HIV, also refers to the amount of atherapeutic agent that increases CD4+ cell counts, increases time toprogression to AIDS, or increases survival time by at least 5%,preferably at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, or more.

The term “amino acid” refers to a molecule containing both an aminogroup and a carboxyl group. Suitable amino acids include, withoutlimitation, both the D- and L-isomers of the 20 common naturallyoccurring amino acids found in peptides (e.g., A, R, N, C, D, Q, E, G,H, I, L, K, M, F, P, S, T, W, Y, V (as known by the one letterabbreviations)) as well as the naturally occurring and non-naturallyoccurring amino acids prepared by organic synthesis or other metabolicroutes.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of a polypeptide (e.g., an HR-1 or HR-2domain) without abolishing or substantially altering itsactivity/secondary structure (alpha-helical structure). An “essential”amino acid residue is a residue that, when altered from the wild-typesequence of the polypeptide, results in abolishing or substantiallyabolishing the polypeptide activity and/or secondary structure.Substantially abolishing is understood as reducing the activity of thepeptide to less than about 30%, less than about 20%, less than about10%, less than about 5% of the wild-type peptide in an appropriate assay(e.g., a syncytia formation assay, a viral fusion assay). The essentialand non-essential amino acid residues of the HR and HR-like domains canreadily be determined by methods well known in the art and are describedherein. In one embodiment, an essential amino acid residue is in the “a”or “d” position of a heptad repeat domain, while non-essential aminoacids may occur in a “b”, “c”, “e”, “f” or “g” position (FIG. 9). Theterm “essential” amino acid residue as used herein, includesconservative substitutions of the essential amino acid. Generally, the“essential” amino acid residues are found at the interacting face of thealpha helix. For example, in the HIV gp41 HR-2 domain the interactingface includes the “a” and “d” position amino acids. (See FIGS. 4A and9). In another embodiment, a modified polypeptide comprises a gp41 HR-1domain having a Leu-556, Leu-565, Val-570, Gly-572, and Arg-579 (Lu, M.,et al., J. Vir, 2001. 75(22); p. 11146-11156).

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. For example, families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Other conserved amino acid substitutions can also occuracross amino acid side chain families, such as when substituting anasparagine for aspartic acid in order to modify the charge of a peptide.Thus, a predicted nonessential amino acid residue in a HR domainpolypeptide, for example, is preferably replaced with another amino acidresidue from the same side chain family or homologues across families(e.g. asparagine for aspartic acid, glutamine for glutamic acid).

As used herein, the terms “identity” or “percent identity”, refers tothe subunit sequence similarity between two polymeric molecules, e.g.,two polynucleotides or two polypeptides. When a subunit position in bothof the two molecules is occupied by the same monomeric subunit, e.g., ifa position in each of two peptides is occupied by serine, then they areidentical at that position. The identity between two sequences is adirect function of the number of matching or identical positions, e.g.,if half (e.g., 5 positions in a polymer 10 subunits in length), of thepositions in two peptide or compound sequences are identical, then thetwo sequences are 50% identical; if 90% of the positions, e.g., 9 of 10are matched, the two sequences share 90% sequence identity. The identitybetween two sequences is a direct function of the number of matching oridentical positions. Thus, if a portion of the reference sequence isdeleted in a particular peptide, that deleted section is not counted forpurposes of calculating sequence identity. Identity is often measuredusing sequence analysis software e.g., BLASTN or BLASTP (available atthe world wide web site (“www”) of the National Center for BiotechnologyInformation (“.ncbi”) of the National Institutes of Health (“.nih”) ofthe U.S. government (“.gov”), in the “Blast” directory (“/BLAST/”). Thedefault parameters for comparing two sequences (e.g., “Blast”-ing twosequences against each other), by BLASTN (for nucleotide sequences) arereward for match=1, penalty for mismatch=−2, open gap=5, extensiongap=2. When using BLASTP for protein sequences, the default parametersare reward for match=0, penalty for mismatch=0, open gap=11, andextension gap=1. Additional, computer programs for determining identityare known in the art.

“Similarity” or “percent similarity” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same or have a specified percentage of amino acid residues,or conservative substitutions thereof, that are the same when comparedand aligned for maximum correspondence, as measured using one of thefollowing sequence comparison algorithms, or by visual inspection. Byway of example, a first polypeptide can be considered similar to anHIV-1 HR-1 domain when the amino acid sequence of the first polypeptideis at least 20%, 50%, 60%, 70%, 75%, 80%, 90%, or even 95% or moreidentical, or conservatively substituted, to a region of the HIV-1 HR-1domain when compared to any sequence of an equal number of amino acidsas the number contained in the first polypeptide as aligned by acomputer similarity program known in the art and described herein.Preferably, the polypeptide region of the first protein and the secondprotein includes one or more conserved amino acid residues.

As used herein, an “antibody” includes any reactive fragment orfragments of antibodies such as Fab molecules, Fab proteins, singlechain polypeptides, or the multi-functional antibodies having bindingaffinity for the antigen. The term includes chimeric antibodies, alteredantibodies, univalent antibodies, bi-specific antibodies, monoclonalantibodies, polyclonal antibodies, human antibodies, and humanizedantibodies. Methods for preparing antibodies are well known in the art.

The symbol

-   -   “        ”        when used as part of a molecular structure refers to a single        bond or a trans or cis double bond.

The term “amino acid side chain” refers to a moiety attached to theα-carbon in an amino acid. For example, the amino acid side chain foralanine is methyl, the amino acid side chain for phenylalanine isphenylmethyl, the amino acid side chain for cysteine is thiomethyl, theamino acid side chain for aspartate is carboxymethyl, the amino acidside chain for tyrosine is 4-hydroxyphenylmethyl, etc. Othernon-naturally occurring amino acid side chains are also included, forexample, those that occur in nature (e.g., an amino acid metabolite) orthose that are made synthetically (e.g., an alpha di-substituted aminoacid).

The term polypeptide encompasses two or more naturally occurring orsynthetic amino acids linked by a covalent bond (e.g., an amide bond).Polypeptides as described herein include full length proteins (e.g.,fully processed proteins) as well as shorter amino acids sequences(e.g., fragments of naturally occurring proteins or syntheticpolypeptide fragments).

The term “halo” refers to any radical of fluorine, chlorine, bromine oriodine. The term “alkyl” refers to a hydrocarbon chain that may be astraight chain or branched chain, containing the indicated number ofcarbon atoms. For example, C₁-C₁₀ indicates that the group may have from1 to 10 (inclusive) carbon atoms in it. In the absence of any numericaldesignation, “alkyl” is a chain (straight or branched) having 1 to 20(inclusive) carbon atoms in it. The term “alkylene” refers to a divalentalkyl (i.e., —R—).

The term “alkenyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon double bonds.The alkenyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkenyl” refers to aC₂-C₈ alkenyl chain. In the absence of any numerical designation,“alkenyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “alkynyl” refers to a hydrocarbon chain that may be a straightchain or branched chain having one or more carbon-carbon triple bonds.The alkynyl moiety contains the indicated number of carbon atoms. Forexample, C₂-C₁₀ indicates that the group may have from 2 to 10(inclusive) carbon atoms in it. The term “lower alkynyl” refers to aC₂-C₈ alkynyl chain. In the absence of any numerical designation,“alkynyl” is a chain (straight or branched) having 2 to 20 (inclusive)carbon atoms in it.

The term “aryl” refers to a 6-carbon monocyclic or 10-carbon bicyclicaromatic ring system wherein 0, 1, 2, 3, or 4 atoms of each ring may besubstituted by a substituent. Examples of aryl groups include phenyl,naphthyl and the like. The term “arylalkyl” or the term “aralkyl” refersto alkyl substituted with an aryl. The term “arylalkoxy” refers to analkoxy substituted with aryl.

The term “cycloalkyl” as employed herein includes saturated andpartially unsaturated cyclic hydrocarbon groups having 3 to 12 carbons,preferably 3 to 8 carbons, and more preferably 3 to 6 carbons, whereinthe cycloalkyl group additionally may be optionally substituted.Preferred cycloalkyl groups include, without limitation, cyclopropyl,cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl,cycloheptyl, and cyclooctyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2, 3,or 4 atoms of each ring may be substituted by a substituent. Examples ofheteroaryl groups include pyridyl, furyl or furanyl, imidazolyl,benzimidazolyl, pyrimidinyl, thiophenyl or thienyl, quinolinyl, indolyl,thiazolyl, and the like. The term “heteroarylalkyl” or the term“heteroaralkyl” refers to an alkyl substituted with a heteroaryl. Theterm “heteroarylalkoxy” refers to an alkoxy substituted with heteroaryl.

The term “heterocyclyl” refers to a nonaromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1, 2 or 3atoms of each ring may be substituted by a substituent. Examples ofheterocyclyl groups include piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, tetrahydrofuranyl, and the like.

The term “substituents” refers to a group “substituted” on an alkyl,cycloalkyl, aryl, heterocyclyl, or heteroaryl group at any atom of thatgroup. Suitable substituents include, without limitation, halo, hydroxy,mercapto, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy,thioalkoxy, aryloxy, amino, alkoxycarbonyl, amido, carboxy,alkanesulfonyl, alkylcarbonyl, and cyano groups.

Ranges provided herein are understood to be shorthand for all of thevalues within the range. For example, a range of 1 to 50 is understoodto include any number, combination of numbers, or sub-range from thegroup consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

Unless specifically stated or obvious from context, as used herein, theterm “or” is understood to be inclusive.

Unless specifically stated or obvious from context, as used herein, theterms “a”, “an”, and “the” are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, theterm “about” is understood as within a range of normal tolerance in theart, for example within 2 standard deviations of the mean. About can beunderstood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%,0.1%, 0.05%, or 0.01% of the stated value.

The recitation of a listing of chemical groups in any definition of avariable herein includes definitions of that variable as any singlegroup or combination of listed groups. The recitation of an embodimentfor a variable or aspect herein includes that embodiment as any singleembodiment or in combination with any other embodiments or portionsthereof.

Any compositions or methods provided herein can be combined with one ormore of any of the other compositions and methods provided herein.

Polypeptides

Described herein are modified peptides which exhibit antiviral activity.It is believed that the modified peptides exhibit antiviral activity viatheir ability to inhibit virus-cell fusion by interfering with viralcoat proteins. The modified peptides of the invention may include astabilized alpha helix heptad repeat domain derived from a virus.Preferably, the alpha helix heptad repeat domain is stabilized withhydrocarbon staples. Suitable viral alpha helix heptad repeat domainscan be derived from any virus with an alpha helical domain (e.g., RSV,influenza, parainfluenza, coronavirus, ebolavirus, HIV) that is directlyor indirectly involved in cell attachment or entry.

While not limited to any theory of operation, the following model isproposed to explain the potent anti-viral activity of the modifiedpolypeptides described herein. When synthesized as stabilized peptides,the modified polypeptides of the invention are potent inhibitors ofviral infection and fusion, likely by virtue of their ability to formcomplexes with viral glycoproteins and interfere with the fusogenicprocess; e.g., during the structural transition of the viral proteinfrom the native structure to the fusogenic state. While not being boundby theory, it is believed the modified peptides gain access to theirrespective binding sites on the viral glycoprotein, and exert adisruptive influence which inhibits fusion of the virus with the cell.The modified polypeptides are particularly useful as a result of theirincreased stability and efficacy.

In a first aspect, the invention is directed to a modified polypeptidehaving a stabilized viral alpha helix heptad repeat domain (e.g., HR-1,HR-2, HR-like or HR-analogs) or active fragment thereof. The modifiedpolypeptide may also comprise a chimera of an HR domain. Suitable viralalpha helix heptad repeat domains can be derived from any virus with analpha helix domain that is directly or indirectly involved in cellattachment or entry.

In another aspect, the invention is directed to a modified polypeptidehaving a stabilized HIV gp41 heptad repeat domain (e.g., heptad repeatdomain 1 or 2 of HIV-1 or HIV-2). The amino acid sequences of heptadrepeat-1 and heptad repeat-2 domains are well known in the art andinclude those represented by SEQ ID NO:2 and SEQ ID NO:1, respectively.In one embodiment, the heptad repeat domain 1 is 30% or more identicalto an amino acid sequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14and forms an alpha helix. Alternatively, the heptad repeat one domain ofthe modified polypeptide may differ by more than 30% as long as theresidues of the interacting face are identical to those of SEQ ID NO:1or 2 or are conservative substitutions thereof. Methods for identifyingthe interacting face residues of the heptad repeat are well known in theart and described herein.

In another embodiment, the heptad repeat domain 2 is 30% or moreidentical to an amino acid sequence of FIG. 4, FIG. 6 or SEQ ID NO:1 andforms an alpha-helix. Alternatively, the heptad repeat two domain of themodified polypeptide may differ by more than 30% as long as the residuesof the interacting face are identical to those of SEQ ID NO:1 or 2 orhave conservative substitutions thereof. Methods for identifying theinteracting face residues of the heptad repeat are well known in the artand described herein.

In one embodiment, the modified polypeptide includes a heptad repeatdomain having the formula: a b c d e f g, wherein a and d arehydrophobic amino acid residues and b, c, e, f and g are any amino acid.Preferably, the formula is repeated in tandem two or more times.

For example, in a further embodiment the heptad repeat domain of themodified polypeptide has the formula: W(a), b, c, W(d), e, f, g, I(a),b, c, Y(d), e, f, g, I(a), b, c, L(d), e, f, g, S(a), b, c, Q(d), e, f,g, N(a), b, c, E(d), e, f, g, L(a), or conservative amino acidsubstitutions thereof and wherein the b, c, e, f and g can be any aminoacid (e.g., residues 3-38 of SEQ ID NO: 44).

In a further, embodiment the heptad repeat domain of the modifiedpolypeptide has the formula: T(g), W(a), b, c, W(d), D(e), R(f), g,I(a), b, c, Y(d), e, f, g, I(a), b, c, L(d), I(e), f, g, a, Q(b), c, d,Q(e), E(f), K(g), a, E(b), c, d, L(e), f, E(g), L(a), or conservativeamino acid substitutions thereof and wherein non-designated amino acidscan be any amino acid (e.g., residues 2-38 of SEQ ID NO: 45).

In another embodiment, the modified polypeptide of the invention is hasthe same amino acid residues, or conservative substitutions thereof, ofthe interacting face of the amino acid sequence of SEQ ID NO:1-14, FIG.5 or FIG. 6. The “interacting face” of the alpha helix are those aminoacid residues which interact with other amino acid residues in a coiledcoil structure. For example, in the HIV gp41 HR-2 domain the interactingface includes the “a” and “d” position amino acids. (See FIGS. 4A and9), while the interacting face of the HIV gp41 HR-1 domain includesamino acids at positions e, g that interact with HR-2 and a, d thatengage in HR1-HR1 interactions (See FIG. 4A). Methods for identifyingheptad repeats and the interacting face residues are well known in theart and described herein.

Preferably the alpha helix heptad repeat domain is stabilized with ahydrocarbon staple (e.g., FIG. 10). Hydrocarbon staples suitable for usewith any of the modified polypeptides are described herein and in U.S.Publication No. 2005/0250680, which is incorporated by reference in itsentirety. Hydrocarbon stapling allows a polypeptide, predisposed to havean alpha-helical secondary structure, to maintain its nativealpha-helical conformation and increase its stability and efficacy. Inone embodiment, the modified polypeptide has at least 10%, 20%, 30%,35%, 40%, 45%, 50%, 60%, 70%, 80%, or 90% or more alpha helicity in anaqueous solution as determined by circular dichroism. Assays fordetermining circular dichroism are known in the art and describedherein.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, which tether significantly enhances the alpha helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3.4 or about7 amino acids). Accordingly, amino acids positioned at i and i+3; i andi+4; or i and i+7 are ideal candidates for chemical modification andcross-linking. Thus, any of the amino acid residues of the modifiedpolypeptides of the invention may be tethered (e.g., cross-linked) inconformity with the above. Suitable tethers are described herein and inU.S. Patent Publication No. 2005/0250680.

In a further embodiment, the hydrocarbon staple(s) is positioned so asto link a first amino acid (i) and a second amino acid (i+3) which is 3amino acids downstream of the first amino acid. In another embodiment,the hydrocarbon staple links a first amino acid (i) and a second aminoacid (i+4) which is 4 amino acids downstream of the first amino acid. Inyet another embodiment, the hydrocarbon staple links a first amino acid(i) and a second amino acid (i+7) which is 7 amino acids downstream ofthe first amino acid.

In yet a further embodiment, the modified polypeptides include a heptadrepeat domain with the sequence:

BTWXEWDXEINNYTSLIHSL (SEQ ID NO: 15),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 16),BTWBXWDRXINNYTSL (SEQ ID NO: 17),BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 18),BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 19),BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 20),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 21),BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 22),BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 23),BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 24),BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 25),BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 26),YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 27),YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 28),YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF (SEQ ID NO: 29),YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 30),YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF (SEQ ID NO: 31),YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 32),YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 33),YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 34),YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 35),YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 36),YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF (SEQ ID NO: 37),YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 38),YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 39),YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF (SEQ ID NO: 40),YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF (SEQ ID NO: 41),BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE (SEQ ID NO: 42), orBTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE (SEQ ID NO: 43);wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine. The modified polypeptides will generally have thestructure of Formula (I), (II) or (III), as described herein.

The invention is also, inter alia, directed to modified polypeptidesfrom other viruses with alpha helical domains that are either directlyor indirectly involved in the attachment and/or fusion of a virus to acell. For example, in one aspect the invention is directed to a modifiedpolypeptide having a stabilized viral alpha helix (e.g., heptad repeatdomain) that is derived from respiratory syncytial virus. The alphahelix may include any alpha helical domain derived from RSV that isinvolved in viral infectivity. Suitable RSV alpha helix domains includethose which are 30% or more identical toYTSVITIELSNIKENKCNGTDAKVKLIKQELDKYKNAVTELQLLMQST (SEQ ID NO:4);FYDPLVFPSDEFDASISQVNEKINQSLAFIRKSDELL (SEQ ID NO:5);SGIAVSKVLHLEGEVNKIKNALLSTNKAVVSLSNGVSVLTSKVLDLKSYINNQ LLPI- (SEQ ID NO:11) or PIINYYDPLVFPSDEFDASISQVNEKINQSLAFIRRSDELLHNVNTGKSTTNIM (SEQ IDNO: 12); and form an alpha-helix.

Alternatively, the heptad repeat analog domain of the modifiedpolypeptide may differ by more than 30% as long as the residues of theinteracting face are identical to those of SEQ ID NOs: 4, 5, 11 and 12or are conservative substitutions thereof. Methods for identifying theinteracting face residues of the heptad repeat analogs are well known inthe art and described herein.

In yet another aspect, the invention is directed to a modifiedpolypeptide having a stabilized viral alpha helix heptad repeat domainthat is derived from a parainfluenza virus. Suitable parainfluenza virusheptad repeat domains include those which are 30% or more identical toALGVATSAQITAAVALVEAKQARSDIEKLKEAIR (SEQ ID NO:6) and form analpha-helix. Alternatively, the heptad repeat domain of the modifiedparainfluenza polypeptide may differ by more than 30% as long as theresidues of the interacting face are identical to those of SEQ ID NO: 6or are conservative substitutions thereof. Methods for identifying theinteracting face residues of the heptad repeat are well known in the artand described herein.

In another aspect, the invention is directed to a modified polypeptidehaving a stabilized viral alpha helix heptad repeat domain derived froma paramyxovirus, orthomyxovirus coronavirus, and a filovirus.

Coronavirus alpha helix heptad repeat domains are known in the art andinclude those which have an amino acid sequence which are 30% or moreidentical to NVLYENQKQIANQFNKAISQIQESLTTTSTALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAE (SEQ ID NO:7) orTSPDVDFGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKY (SEQ ID NO:8) and forman alpha-helix. Alternatively, the heptad repeat domain of the modifiedcoronavirus polypeptide may differ by more than 30% as long as theresidues of the interacting face are identical to those of SEQ ID NOs: 7and 8 or are conservative substitutions thereof. Methods for identifyingthe interacting face residues of the heptad repeat are well known in theart and described herein.

Similarly, filovirus alpha helix heptad repeat domains are known in theart and include those that are 30% or more identical toDGLICGLRQLANETTQALQLFLRATTELRTFSILNRKAIDFLL (SEQ ID NO:9) orDWTKNITDKIDQIIHDFVDKTLPD (SEQ ID NO:10) and form an alpha-helix.Alternatively, the heptad repeat domain of the modified filoviruspolypeptide may differ by more than 30% as long as the residues of theinteracting face are identical to those of SEQ ID NO: 10 or areconservative substitutions thereof. Methods for identifying theinteracting face residues of the heptad repeat are well known in the artand described herein.

Influenza heptad repeat domains are also known in the art. For example,a heptad repeat domain in Influenza A Virus (strain A/Aichi/2/68) occursat residues 379-436, 387-453, and 380-456. Similarly, residues 383-471were shown by Carr and Kim to be an extended coiled coil when underacidic pH (Carr and Kim, 1993, Cell 73: 823-832).

The modified polypeptides of the invention will generally include thestructure of Formula (I), (II) or (III) provided below.

Any of the modified polypeptides described herein can be present in acomposition (e.g., pharmaceutical composition) or kit. In someembodiments of the invention, the composition or kit comprises two ormore modified polypeptides. For example, the composition may include twoor more modified polypeptides having a stabilized HIV gp41 heptad repeatdomain.

For clarity of discussion, the invention will be further describedprimarily for HR-1 and HR-2 modified polypeptides of HIV. However, theprinciples may be analogously applied to other viruses, both envelopedand nonenveloped, and to other non-viral organisms. As used herein theterm “heptad repeat” includes HR-2 and HR-1 peptides.

HR-2 and HR-2-Peptides

The modified polypeptides of the invention include the HR-2 peptides(SEQ ID NO:1 and 13) which corresponds to amino acid residues 638 to 673and 626 and 662 respectively of gp160 from the HIV-1 (SEQ ID NO:13), andhas the 36 and 37 amino acid sequences, respectively, of (reading fromamino to carboxy terminus):

(SEQ ID NO: 1) YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF and (SEQ ID NO: 13)MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE.

Other useful HR-2 polypeptides for use with the current invention aredescribed in U.S. Pat. No. 7,273,614, which is incorporated herein byreference in its entirety.

In addition to the use of full-length HR-2 (SEQ ID NO:1 and 13) 36 and37-mers and the corresponding sequences and variants thereof found inthe diversity of HIV-1 strains and mutants, the peptides of theinvention may include truncations of the HR-2 (SEQ ID NO: 1 and 13)peptide, gp41 polypeptide sequences that flank the HR-2 domain (ie.immediately upstream or downstream sequences), or chimeras which exhibitantifusogenic activity and antiviral activity. Truncations of HR-2 (SEQID NO:1 and 13) peptides may comprise peptides of between 3 and 36 aminoacid residues, as shown in FIGS. 5 and 6. Peptide sequences in thisfigure are listed from amino (left) to carboxy (right) terminus.

The modified peptides of the invention also include HR-2-like peptides.“HR-2-like” or “heptad repeat-like”, as used herein, refers tofull-length and truncated and chimeric HR-2 polypeptides which containone or more amino acid substitutions, insertions and/or deletions aswell as peptide sequences identified or recognized by homologysearching. Representative HR-2 like polypeptides include thoseillustrated in FIG. 5 or FIG. 6. The modified HR-2-like peptides of theinvention may exhibit antifusogenic or antiviral activity. In oneembodiment, the heptad repeat domain 2 is 30% or more identical to anamino acid sequence of FIG. 5, FIG. 6, SEQ ID NO:1 or SEQ ID NO:13 andform an alpha-helix. Alternatively, the heptad repeat domain 2 of themodified polypeptide may differ by more than 30% as long as the residuesof the interacting face are identical to those of FIG. 5, FIG. 6, SEQ IDNO:1 or SEQ ID NO:13 or are conservative substitutions thereof. Methodsfor identifying the interacting face residues of the heptad repeat arewell known in the art and described herein.

HIV-1 and HIV-2 enveloped proteins are structurally distinct, but thereexists a striking amino acid conservation within the HR-2 regions ofHIV-1 and HIV-2. The amino acid conservation is of a periodic nature,suggesting some conservation of structure and/or function. Therefore,one possible class of amino acid substitutions would include those aminoacid changes which are predicted to stabilize the structure of the HR-2peptides of the invention. Utilizing the HR-2 and HR-2 analog sequencesdescribed herein, the skilled artisan can readily compile HR-2 consensussequences and ascertain from these, conserved amino acid residues whichwould represent preferred amino acid substitutions.

The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions consist of replacing one ormore amino acids of the HR-2 (SEQ ID NO:1 or 13) peptide sequence withamino acids of similar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to asparticacid (D), aspartic acid (D) to asparagine (N), and glutamic acid (E) toglutamine (Q) amino acid substitution. Non-conserved substitutionsconsist of replacing one or more amino acids of the HR-2 peptidesequence with amino acids possessing dissimilar charge, size, and/orhydrophobicity characteristics, such as, for example, a glutamic acid(E) to valine (V) substitution.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the full length or truncated HR-2 peptides, aswell as at a position internal to the peptide. Such insertions willgenerally range from 2 to 15 amino acids in length. It is contemplatedthat insertions made at either the carboxy or amino terminus of thepeptide of interest may be of a broader size range, with about 2 toabout 50 amino acids being preferred. One or more such insertions may beintroduced into the full-length (SEQ ID NO:1 or 13) or truncated HR-2polypeptides as long as such insertions result in modified peptides thatexhibit antifusogenic or antiviral activity.

Preferred amino or carboxy terminal insertions are peptides ranging fromabout 2 to about 50 amino acid residues in length, corresponding to gp41protein regions either amino to or carboxy to the actual HR-2 gp41 aminoacid sequence, respectively. Thus, a preferred amino terminal or carboxyterminal amino acid insertion would contain gp41 amino acid sequencesfound immediately amino to or carboxy to the HR-2 region of the gp41protein.

Deletions of full-length (SEQ ID NO:1 or 13) or truncated HR-2polypeptides are also within the scope of the invention. Such deletionsconsist of the removal of one or more amino acids from the HR-2 orHR-2-like peptide sequence, with the lower limit length of the resultingpeptide sequence being 4 to 6 amino acids. Such deletions may involve asingle contiguous or greater than one discrete portion of the peptidesequences. One or more such deletions may be introduced into full-length(SEQ ID NO: 1 or 13) or truncated HR-2 polypeptides, as long as suchdeletions result in peptides which may still exhibit antifusogenic orantiviral activity.

HR-1 and HR-1-Peptides

Further, the modified peptides of the invention include peptides havingamino acid sequences corresponding to HR-1 analogs. HR-1 includes 38-and 51-amino acid peptides which exhibits potent antiviral activity, andcorresponds to residues 553 to 590 and 542-592, respectively, of HIV-1transmembrane (TM) gp41 protein, as shown below:

(SEQ ID NO: 2) NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLQDQ or (SEQ ID NO: 14RQLLSGIVQQQ NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERY LQDQQL.

In addition to the full-length HR-1 38-mer, the modified peptides of theinvention include truncations of the HR-1 peptide which exhibitantifusogenic activity or antiviral activity. Truncations of HR-1peptides can be made in a similar manner as those exemplified for theHR-2 peptides in FIG. 5 and FIG. 6.

The modified peptides of the invention also include HR-1-like peptides.“HR-1-like” or “heptad-repeat like”, as used herein, refers tofull-length and truncated HR-1 polypeptides which contain one or moreamino acid substitutions, insertions and/or deletions and exhibitingantifusogenic or antiviral activity. In one embodiment, the heptadrepeat domain 1 is 30% or more identical to an amino acid sequence ofSEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and form an alpha-helix.Alternatively, the heptad repeat domain 1 of the modified polypeptidemay differ by more than 30% as long as the residues of the interactingface are identical to those of SEQ ID NOs 2, 3 or 14 or are conservativesubstitutions thereof. Methods for identifying the interacting faceresidues of the heptad repeat are well known in the art and describedherein.

HIV-1 and HIV-2 enveloped proteins are structurally distinct, but thereexists a striking amino acid conservation within the HR-1-correspondingregions of HIV-1 and HIV-2. The amino acid conservation is of a periodicnature, suggesting some conservation of structure and/or function.Therefore, one possible class of amino acid substitutions would includethose amino acid changes which are predicted to stabilize the structureof the HR-1 peptides of the invention. Utilizing the HR-1 and HR-1analog sequences described herein, the skilled artisan can readilycompile HR-1 consensus sequences and ascertain from these, conservedamino acid residues which would represent preferred amino acidsubstitutions.

The amino acid substitutions may be of a conserved or non-conservednature. Conserved amino acid substitutions consist of replacing one ormore amino acids of the HR-1 peptide sequence with amino acids ofsimilar charge, size, and/or hydrophobicity characteristics, such as,for example, a glutamic acid (E) to aspartic acid (D), aspartic acid (D)to asparagine (N), and glutamic acid (E) to glutamine (Q) amino acidsubstitution. Non-conserved substitutions consist of replacing one ormore amino acids of the HR-1 peptide sequence with amino acidspossessing dissimilar charge, size, and/or hydrophobicitycharacteristics, such as, for example, a glutamic acid (E) to valine (V)substitution.

Amino acid insertions may consist of single amino acid residues orstretches of residues. The insertions may be made at the carboxy oramino terminal end of the full-length or truncated HR-1 peptides, aswell as at a position internal to the peptide. Such insertions willgenerally range from 2 to 15 amino acids in length. It is contemplatedthat insertions made at either the carboxy or amino terminus of thepeptide of interest may be of a broader size range, with about 2 toabout 50 amino acids being preferred. One or more such insertions may beintroduced into full-length or truncated HR-1 polypeptides, as long assuch insertions result in modified peptides which may still exhibitantifusogenic or antiviral activity.

Preferred amino or carboxy terminal insertions are peptides ranging fromabout 2 to about 50 amino acid residues in length, corresponding to gp41protein regions either amino to or carboxy to the actual HR-1 gp41 aminoacid sequence, respectively. Thus, a preferred amino terminal or carboxyterminal amino acid insertion would contain gp41 amino acid sequencesfound immediately amino to or carboxy to the HR-1 region of the gp41protein.

Deletions of full-length or truncated HR-1 polypeptides are also withinthe scope of the invention. Such deletions consist of the removal of oneor more amino acids from the HR-1 or HR-1-like peptide sequence, withthe lower limit length of the resulting peptide sequence being 4 to 6amino acids. Such deletions may involve a single contiguous or greaterthan one discrete portion of the peptide sequences. One or more suchdeletions may be introduced into full-length or truncated HR-1polypeptides, as long as such deletions result in peptides which maystill exhibit antifusogenic or antiviral activity

HR-1 and HR-2 Analogs

Peptides corresponding to analogs of the full-length and truncated HR-1and HR-2 polypeptides, described, above, may be found in other viruses.The term “HR-1 and HR-2-analogs”, as used herein, refers to a peptidewhich is recognized or identified as having a heptad repeat-analogdomain in a non-HIV virus. Methods for identifying heptad repeat-analogpolypeptides are known in the art, for example, bioinformatics programsbased on pairwise residue correlations (e.g., on the world wide web at:ch.embnet.org/software/COILS_form.html), which have the ability torecognize coiled coils from protein sequences and model their structures(See Lupas, A., et al. Science 1991. 252(5009); p. 1162-1164, which isherein incorporated by reference). Further, such modified peptidesexhibit antifusogenic or antiviral activity.

Such HR-2 and HR-1 analogs may, for example, correspond to peptidesequences present in transmembrane proteins of other enveloped viruses.Such peptides may exhibit antifusogenic activity or antiviral activity.

HR-2 analogs are peptides whose amino acid sequences are comprised ofthe amino acid sequences of peptide regions of, for example, otherviruses that correspond to the gp41 peptide region from which HR-2 (SEQID NO: 1) was derived. Such viruses may include, but are not limited to,other HIV-1 isolates, HIV-2 isolates, SIV isolates, influenza,parainfluenza virus, coronavirus, RSV, etc.

HR-1 analogs are peptides whose amino acid sequences are comprised ofthe amino acid sequences of peptide regions of, for example, otherviruses that correspond to the gp41 peptide region from which HR-1 (SEQID NO: 2) was derived. Such viruses may include, but are not limited to,other HIV-1 isolates HIV-2 isolates, SIV isolates, parainfluenza virus,coronavirus, RSV, etc.

HR-1 and HR-2 analogs or other heptad repeat polypeptides includepeptides whose amino acid sequences are comprised of the amino acidsequences of peptide regions of, for example, other viruses thatcorrespond to the gp41 peptide region from which HR-1 (SEQ ID NO: 2 orSEQ ID NO:3) and HR-2 (SEQ ID NO:1) were derived. These polypeptidesinclude:

RSV heptad repeat domains which are 30% or more identical to (SEQ IDNO:4), (SEQ ID NO:5), (SEQ ID NO: 11), or (SEQ ID NO: 12) and form analpha-helix.

Parainfluenza virus heptad repeat domains which are 30% or moreidentical to (SEQ ID NO:6) and form an alpha-helix.

Coronavirus alpha helix heptad repeat domains which are 30% or moreidentical to (SEQ ID NO:7) or (SEQ ID NO:8) and form an alpha-helix.

Filovirus alpha helix heptad repeat domains which are 30% or moreidentical to (SEQ ID NO:9) or (SEQ ID NO:10) and form an alpha-helix.

The modified polypeptides of the invention also contemplate the use ofinfluenza virus heptad repeat domains.

Alternatively, the heptad repeat domains of the modified polypeptidesmay differ by more than 30% as long as the residues of the interactingface are identical to those of the interacting face of the referencesequence or are conservative substitutions thereof. Methods foridentifying the interacting face residues of the heptad repeat are wellknown in the art and described herein.

Heptad repeats or heptad repeat-analogs are recognized or identified,for example, by utilizing computer-assisted search strategies known inthe art. For example, bioinformatics programs based on pairwise residuecorrelations (e.g., on the world wide web at:ch.embnet.org/software/COILS_form.html), which have the ability torecognize coiled coils from protein sequences and model their structures(See Lupas, A., et al. Science 1991. 252(5009); p. 1162-1164, and U.S.Pat. No. 7,273,614 both of which are herein incorporated by reference inits entirety. The search strategy can identify additional peptideregions which are predicted to have structural and/or amino acidsequence features similar to those of HR-1 and/or HR-2.

Stabilization of Heptad Repeat Polypeptides

The modified polypeptides of the present invention have stabilized(e.g., cross-linked) alpha helical domains. Preferable the polypeptidesare hydrocarbon-stapled. Hydrocarbon stapling is described in U.S.Patent Publication No. 2005/0250680, which is herein incorporated byreference in its entirety.

The peptide α-helix participates in critically important proteininteractions by presenting specific amino acid residues in an orderedand precise arrangement over a relatively large contact surface area(Chittenden, T., et al., Embo Journal, 1995. 14(22): p. 5589-5596;Kussie, P. H., et al. Science, 1996. 274(5289): p. 948-953; Ellenberger,T. E., et al., Cell, 1992. 71(7): p. 1223-1237). Alpha-helical domainsare frequently stabilized by scaffold sequences in the remainder of theprotein, which facilitate the preorganization of α-helical structure.When taken out of context, α-helical peptide motifs can unfold, leadingto loss of biological activity. Critical challenges is developingα-helical peptides include maintaining their natural α-helical structureand preparing peptides that can resist proteolytic, acid and thermaldegradation, and thereby remain intact in vivo.

Hydrocarbon stapling, refers to a process for stably cross-linking apolypeptide via at least two amino acids that helps to conformationallybestow the native secondary structure of that polypeptide. Hydrocarbonstapling allows a polypeptide, predisposed to have an alpha-helicalsecondary structure, to maintain its native alpha-helical conformation.This secondary structure increases resistance of the polypeptide toproteolytic cleavage and heat, and also may increase hydrophobicity.Accordingly, the hydrocarbon stapled (cross-linked) polypeptidesdescribed herein have improved biological activity relative to acorresponding non-hydrocarbon stapled (uncrosslinked) polypeptide. Forexample the cross-linked polypeptide can include an alpha-helical domainof an HIV polypeptide (e.g., HR-1/HR-2 domain), which can interfere withHIV attachment, fusion with, and infection of a cell. In some instances,the cross-linked polypeptide can be used to inhibit virus entry into acell. The cross-linked polypeptides described herein can be usedtherapeutically, e.g., to treat HIV.

The hydrocarbon stapled polypeptides include a tether (linkage) betweentwo amino acids, which tether significantly enhances the alpha helicalsecondary structure of the polypeptide. Generally, the tether extendsacross the length of one or two helical turns (i.e., about 3.4 or about7 amino acids). Accordingly, amino acids positioned at i and i+3; i andi+4; or i and i+7 are ideal candidates for chemical modification andcross-linking. Thus, for example, where a peptide has the sequence . . .X1, X2, X3, X4, X5, X6, X7, X8, X9 . . . , cross-links between X1 andX4, or between X1 and X5, or between X1 and X8 are useful as arecross-links between X2 and X5, or between X2 and X6, or between X2 andX9, etc. The use of multiple cross-links (e.g., 2, 3, 4 or more) hasalso been achieved, compounding the benefits of individual stapledadducts (e.g. improved helicity and activity; improved helicity andthermal stability; improved helicity and acid stability). Thus, theinvention encompasses the incorporation of more than one crosslinkwithin the polypeptide sequence to either further stabilize the sequenceor facilitate the structural stabilization, proteolytic resistance,thermal stability, acid stability, and biological activity enhancementof longer polypeptide stretches.

In one embodiment, the modified polypeptides of the invention have theformula

wherein;

-   each R₁ and R₂ are independently H or a C₁ to C₁₀ alkyl, alkenyl,    alkynyl, arylalkyl, cycloalkylalkyl, heteroarylalkyl, or    heterocyclylalkyl;    R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n); each of which is    substituted with 0-6 R₅;-   R₄ is alkyl, alkenyl, or alkynyl;-   R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, a    fluorescent moiety, or a radioisotope;-   K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

-   R₆ is H, alkyl, or a therapeutic agent;-   n is an integer from 1-4;-   x is an integer from 2-10;-   each y is independently an integer from 0-100;-   z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and    each Xaa is independently an amino acid. The modified polypeptides    may includes an amino acid sequence which forms an alpha-helix and    is 30% or more identical to an amino acid sequence of SEQ ID    NO:1-14, FIG. 5, FIG. 6,

BTWXEWDXEINNYTSLIHSLB (SEQ ID NO: 15),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 16),BTWBXWDRXINNYTSL (SEQ ID NO: 17),BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 18),BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 19),BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 20),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 21),BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 22),BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 23),BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 24),BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 25),BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 26),YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 27),YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 28),YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF (SEQ ID NO: 29),YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 30),YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF (SEQ ID NO: 31),YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 32),YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 33),YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 34),YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 35),YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 36),YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF (SEQ ID NO: 37),YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 38),YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 39),YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF (SEQ ID NO: 40),YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF (SEQ ID NO: 41),BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE (SEQ ID NO: 42), orBTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE (SEQ ID NO: 43);wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine.

The tether can include an alkyl, alkenyl, or alkynyl moiety (e.g., C₅,C₈ or C₁₁ alkyl or a C₅, C₈ or C₁₁ alkenyl, or C₅, C₈ or C₁₁ alkynyl).The tethered amino acid can be alpha disubstituted (e.g., C₁-C₃ ormethyl).

In some instances, x is 2, 3, or 6.

In some instances, each y is independently an integer between 3 and 15.

In some instances each y is independently an integer between 1 and 15.

In some instances, R₁ and R₂ are each independently H or C₁-C₆ alkyl.

In some instances, R₁ and R₂ are each independently C₁-C₃ alkyl.

In some instances, at least one of R₁ and R₂ are methyl. For example R₁and R₂ are both methyl.

In some instances R₃ is alkyl (e.g., C₈ alkyl) and x is 3.

In some instances, R₃ is C₁₁ alkyl and x is 6.

In some instances, R₃ is alkenyl (e.g., C₈ alkenyl) and x is 3.

In some instances x is 6 and R₃ is C₁₁ alkenyl.

In some instances, R₃ is a straight chain alkyl, alkenyl, or alkynyl.

In some instances R₃ is —CH₂—CH₂—CH₂—CH═CH—CH₂—CH₂—CH₂—.

In certain embodiments the two alpha, alpha disubstituted stereocentersare both in the R configuration or S configuration (e.g., i, i+4cross-link), or one stereocenter is R and the other is S (e.g., i, i+7cross-link). Thus, where formula I is depicted as

the C′ and C″ disubstituted stereocenters can both be in the Rconfiguration or they can both be in the S configuration, for examplewhen X is 3. When x is 6, the C′ disubstituted stereocenter is in the Rconfiguration and the C″ disubstituted stereocenter is in the Sconfiguration. The R₃ double bond may be in the E or Z stereochemicalconfiguration.

In some instances R₃ is [R₄—K—R₄]_(n); and R4 is a straight chain alkyl,alkenyl, or alkynyl.

In some embodiments the modified polypeptide comprises at least 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50,or more contiguous amino acids of a heptad repeat or heptad repeat likedomain, e.g., a HIV-1 HR-1 or HR-2 domain. Each [Xaa]y is a peptide thatcan independently comprise at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 25 or more contiguous amino acids of a heptadrepeat or heptad repeat like domain, e.g., a HIV-1 HR-1 or HR-2 domain,e.g., a polypeptide depicted in any of FIGS. 5 and 6. [Xaa]_(R) is apeptide that can comprise 3 or 6 contiguous amino acids of acids of aheptad repeat or heptad repeat like domain, e.g., a HIV-1 HR-1 domain orHR-2, e.g., a polypeptide having the amino acid sequence of SEQ IDNO:1-14 or FIG. 5 or 6.

The modified polypeptide can comprise 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 contiguous aminoacids of acids of a heptad repeat or heptad repeat like domain, e.g., aHIV-1 HR-1 domain or HR-2, e.g., a polypeptide having the amino acidsequence of SEQ ID NO:1-14 or FIG. 5 or 6, wherein two amino acids thatare separated by two, three, or six amino acids are replaced by aminoacid substitutes that are linked via R₃. Thus, at least two amino acidscan be replaced by tethered amino acids or tethered amino acidsubstitutes. Thus, where formula (I) is depicted as

[Xaa]_(y′) and [Xaa]_(y″) can each comprise contiguous polypeptidesequences from the same or different heptad repeat or heptad repeat likedomains.

The invention features cross-linked polypeptides comprising 10 (11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50or more) contiguous amino acids of a heptad repeat or heptad repeat likedomain, e.g., a HIV-1 HR-1 domain or HR-2, e.g., a polypeptide havingthe amino acid sequence of SEQ ID NO:1-14 or FIG. 5 or 6, wherein thealpha carbons of two amino acids that are separated by two, three, orsix amino acids are linked via R₃, one of the two alpha carbons issubstituted by R₁ and the other is substituted by R₂ and each is linkedvia peptide bonds to additional amino acids.

In another embodiment, the modified polypeptides of the invention havethe formula (II),

wherein

-   each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl,    arylalkyl, cycloalkylalkyl; heteroarylalkyl; or heterocyclylalkyl;-   each n is independently an integer from 1-15;-   x is 2, 3, or 6-   each y is independently an integer from 0-100;-   z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10);-   each Xaa is independently an amino acid.

The polypeptide forms and alpha-helix and includes an amino acidsequence which is about 30% or more identical to an amino acid sequenceof SEQ ID NO:1-14, FIG. 5, FIG. 6 or

the modified polypeptides include a heptad repeat domain with thesequence:

BTWXEWDXEINNYTSLIHSL (SEQ ID NO: 15),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 16),BTWBXWDRXINNYTSL (SEQ ID NO: 17),BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 18),BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 19),BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 20),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 21),BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 22),BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 23),BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 24),BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 25),BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 26),YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 27),YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 28),YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF (SEQ ID NO: 29),YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 30),YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF (SEQ ID NO: 31),YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 32),YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 33),YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 34),YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 35),YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 36),YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF (SEQ ID NO: 37),YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 38),YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 39),YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF (SEQ ID NO: 40),YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF (SEQ ID NO: 41),BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE (SEQ ID NO: 42), orBTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE (SEQ ID NO: 43);wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine.

In still another embodiment, the modified polypeptides of the inventionhave the formula (III),

wherein;

-   each R₁ and R₂ are independently H, alkyl, alkenyl, alkynyl,    arylalkyl, cycloalkylalkyl, heteroarylalkyl, or heterocyclylalkyl;-   R₃ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or a naturally    occurring amino acid side chain; each of which is substituted with    0-6 R₅;-   R₄ is alkyl, alkenyl, or alkynyl;-   R₅ is halo, alkyl, OR₆, N(R₆)₂, SR₆, SOR₆, SO₂R₆, CO₂R₆, R₆, a    fluorescent moiety, or a radioisotope;-   K is O, S, SO, SO₂, CO, CO₂, CONR₆, or

-   R₆ is H, alkyl, or a therapeutic agent;-   R₇ is alkyl, alkenyl, alkynyl; [R₄—K—R₄]_(n) or an naturally    occurring amino acid side chain; each of which is substituted with    0-6 R₅;-   n is an integer from 1-4;-   x is an integer from 2-10;-   each y is independently an integer from 0-100;-   z is an integer from 1-10 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10); and-   each Xaa is independently an amino acid;

The polypeptide forms and alpha-helix and includes an amino acidsequence which is about 30% or more identical to an amino acid sequenceof SEQ ID NO:1-14, FIG. 5, FIG. 6 or

the modified polypeptides include a heptad repeat domain with thesequence:

BTWXEWDXEINNYTSLIHSL (SEQ ID NO: 15),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 16),BTWBXWDRXINNYTSL (SEQ ID NO: 17),BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 18),BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 19),BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 20),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 21),BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 22),BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 23),BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 24),BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 25),BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 26),YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 27),YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 28),YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF (SEQ ID NO: 29),YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 30),YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF (SEQ ID NO: 31),YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 32),YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 33),YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 34),YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 35),YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 36),YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF (SEQ ID NO: 37),YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 38),YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 39),YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF (SEQ ID NO: 40),YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF (SEQ ID NO: 41),BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE (SEQ ID NO: 40), orBTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE (SEQ ID NO: 41);wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine.

While hydrocarbon tethers have been described, other tethers are alsoenvisioned. For example, the tether can include one or more of an ether,thioether, ester, amine, or amide moiety. In some cases, a naturallyoccurring amino acid side chain can be incorporated into the tether. Forexample, a tether can be coupled with a functional group such as thehydroxyl in serine, the thiol in cysteine, the primary amine in lysine,the acid in aspartate or glutamate, or the amide in asparagine orglutamine. Accordingly, it is possible to create a tether usingnaturally occurring amino acids rather than using a tether that is madeby coupling two non-naturally occurring amino acids. It is also possibleto use a single non-naturally occurring amino acid together with anaturally occurring amino acid.

It is further envisioned that the length of the tether can be varied.For instance, a shorter length of tether can be used where it isdesirable to provide a relatively high degree of constraint on thesecondary alpha-helical structure, whereas, in some instances, it isdesirable to provide less constraint on the secondary alpha-helicalstructure, and thus a longer tether may be desired.

Additionally, while examples of tethers spanning from amino acids i toi+3, i to i+4; and i to i+7 have been described in order to provide atether that is primarily on a single face of the alpha helix, thetethers can be synthesized to span any combinations of numbers of aminoacids.

As can be appreciated by the skilled artisan, methods of synthesizingthe compounds of the described herein will be evident to those ofordinary skill in the art. Additionally, the various synthetic steps maybe performed in an alternate sequence or order to give the desiredcompounds. Synthetic chemistry transformations and protecting groupmethodologies (protection and deprotection) useful in synthesizing thecompounds described herein are known in the art and include, forexample, those such as described in R. Larock, Comprehensive OrganicTransformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts,Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons(1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed.,Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995), and subsequent editions thereof.

Synthesis of Peptides

The peptides of this invention can be made by chemical synthesismethods, which are well known to the ordinarily skilled artisan anddescribed herein. See, for example, Fields et al., Chapter 3 inSynthetic Peptides: A User's Guide, ed. Grant, W. H. Freeman & Co., NewYork, N.Y., 1992, p. 77. Hence, peptides can be synthesized using theautomated Merrifield techniques of solid phase synthesis with thealpha-NH₂ protected by either t-Boc or F-moc chemistry using side chainprotected amino acids on, for example, an Applied Biosystems PeptideSynthesizer Model 430A or 431 or the AAPPTEC multichannel synthesizerAPEX 396.

One manner of making of the peptides described herein is using solidphase peptide synthesis (SPPS). The C-terminal amino acid is attached toa cross-linked polystyrene resin via an acid labile bond with a linkermolecule. This resin is insoluble in the solvents used for synthesis,making it relatively simple and fast to wash away excess reagents andby-products. The N-terminus is protected with the Fmoc group, which isstable in acid, but removable by base. Any side chain functional groupsare protected with base stable, acid labile groups.

Longer peptides could be made by conjoining individual syntheticpeptides using native chemical ligation. Alternatively, the longersynthetic peptides can be synthesized by well known recombinant DNAtechniques. Such techniques are provided in well-known standard manualswith detailed protocols. To construct a gene encoding a peptide of thisinvention, the amino acid sequence is reverse translated to obtain anucleic acid sequence encoding the amino acid sequence, preferably withcodons that are optimum for the organism in which the gene is to beexpressed. Next, a synthetic gene is made, typically by synthesizingoligonucleotides which encode the peptide and any regulatory elements,if necessary. The synthetic gene is inserted in a suitable cloningvector and transfected into a host cell. Furthermore, the host cell isengineered so as to be able to incorporate the non-natural amino acidsfor the hydrocarbon staple. The peptide is then expressed under suitableconditions appropriate for the selected expression system and host. SeeLiu et al. Proc. Nat. Acad. Sci (USA), 94:10092-10097 (1997). Thepeptide is purified and characterized by standard methods.

The peptides can be made in a high-throughput, combinatorial fashion,e.g., using a high-throughput polychannel combinatorial synthesizeravailable from Advanced Chemtech.

Assaying Anti-Viral Activity

Described herein, are methods for evaluating the ability of a compound,such as the peptides of the invention, to inhibit membrane fusion and/orexhibit anti-viral activity both in vitro and in vivo. Specifically,such assays are described below and in Examples 4 and 5. Additionalassays for evaluating anti-vial activity are well known to those withordinary skill in the art.

The antiviral activity exhibited by the peptides of the invention may bemeasured, for example, by easily performed in vitro assays, such asthose described herein and known by those of ordinary skill in the art,which can test the peptides' ability to inhibit syncytia formation, ortheir ability to inhibit infection by cell-free virus (Madani, N., etal., Journal of Virology, 2007. 81(2): p. 532-538; Si, Z. H., M.Cayabyab, and J. Sodroski, Journal of Virology, 2001. 75(9): p.4208-4218; Si, Z. H., et al., PNAS USA, 2004. 101(14): p. 5036-5041).

Using these assays, such parameters as the relative antiviral activityof the peptides exhibit against a given strain of virus and/or thestrain specific inhibitory activity of the peptide can be determined.

Assays to test a peptide's antiviral capabilities are contemplated withthe present invention. Taking HIV as an example, a reverse transcriptase(RT) assay may be utilized to test the peptides' ability to inhibitinfection of CD-4⁺ cells by cell-free HIV. Such an assay may compriseculturing an appropriate concentration (i.e., Tissue Culture InfectiousDose 50) of virus and CD-4+ cells in the presence of the peptide to betested. Culture conditions well known to those in the art are used. Arange of peptide concentrations may be used, in addition to a controlculture wherein no peptide has been added. After incubation for anappropriate period (e.g., 7 days) of culturing, a cell-free supernatantis prepared, using standard procedures, and tested for the present of RTactivity as a measure of successful infection. The RT activity may betested using standard techniques such as those described by, forexample, Goff et al. (Goff, S. et al., 1981, J. Virol. 38:239-248)and/or Willey et al. (Willey, R. et al., 1988, J. Virol. 62:139-147).These references are incorporated herein by reference in their entirety.

Standard methods which are well-known to those of skill in the art maybe utilized for assaying non-retroviral activity. See, for example,Pringle et al. (Pringle, C. R. et al., 1985, J. Medical Virology17:377-386) for a discussion of respiratory syncytial virus andparainfluenza virus activity assay techniques. Further, see, forexample, “Zinsser Microbiology”, 1988, Joklik, W. K. et al., eds.,Appleton & Lange, Norwalk, Conn., 19th ed., for a general review of suchtechniques. These references are incorporated by reference herein intheir entirety.

It is known that HIV positive patients who respond to initial treatmentwith enfuvirtide, may ultimately develop a viral rebound that typicallyoccurs within a maximum of 80 weeks. Resistance to enfuvirtide derivesfrom mutations within the HR-1 region of gp41, although some geneticchanges are found in the HR-2 domain (Xu, L., et al., AntimicrobialAgents and Chemotherapy, 2005. 49(3): p. 1113-1119; Perez-Alvarez, L.,et al. Journal of Medical Virology, 2006. 78(2): p. 141-147). Thesemutations, such as I37V, V38A/E/M, Q39R, Q40H, N42T/Q/H, N43D/Q, areonly found in enfuvirtide-experienced patients (Poveda, E., et al.,Journal of Medical Virology, 2004. 74(1): p. 21-28; Melby, T., et al.,Aids Research and Human Retroviruses, 2006. 22(5): p. 375-385; Sista, P.R., et al., Aids, 2004. 18(13): p. 1787-1794; Wei, X. P., et al.,Antimicrobial Agents and Chemotherapy, 2002. 46(6): p. 1896-1905).

Modified polypeptides of the invention can be developed which are ableto inhibit these enfuvirtide resistant HIV strains. One suitable methodfor assessing the ability of the modified polypeptides to treat theseenfuvirtide resistant HIV strains is a five-helix bundle assay asdescribed in Root, M. J., M. S. Kay, and P. S. Kim, Science, 2001.291(5505): p. 884-888.

Briefly, the five-helix bundle assay would include polypeptides thatincorporate resistance mutations. FITC-labeled SAH-gp41 compounds canthen be screened against these mutant five-helix bundle proteins todetermine if any native SAH-gp41 compounds retain activity despite HRdomain mutations. The FITC labeled mutants SAH-gp41 (mSAH-gp41)compounds can be screened for binding affinity to mutant five-helixbundle proteins and for suppression of HIV infectivity using primaryresistance strains.

In another aspect, the modified polypeptides of the invention can beused to monitor the evolution of resistance in HIV isolates. To explorethe evolution of potential resistance to SAH-gp41 compounds, HIV strainscan be incubated in the presence of increasing concentrations of leadSAH-gp41 compounds in a cell culture. Resistant strains can be genotypedto monitor the evolution of resistance. (See Dwyer et al. Proc. Natl.Acad. Sci., 104:12772 (2007)). Because resistance to one modifiedpolypeptide of the invention may not affect susceptibility to othervariants, (Ray, N., et al., Journal of Virology, 2007. 81(7): p.3240-3250) it is contemplated that treatment may include a combinationof different SAH-gp41 polypeptides that are able to treat resistantstrains of HIV.

In vivo assays may also be utilized to test, for example, the antiviralactivity of the peptides of the invention. To test for anti-HIVactivity, for example, the in vivo model described in Barnett et al.(Barnett, S. W. et al., 1994, Science 266:642-646) may be used.

Additionally, anti-RSV activity can be assayed in vitro using the RSVplaque assay and in vivo via well known mouse models (Kong et al.,Virology J. 2(1):3 (2005). For example, RSV can be administeredintranasally to mice of various inbred strains. Virus replicates inlungs of all strains, but the highest titers are obtained in P/N, C57L/Nand DBA/2N mice. Infection of BALB/c mice produces an asymptomaticbronchiolitis characterized by lymphocytic infiltrates and pulmonaryvirus titers of 104 to 10⁵ pfu/g of lung tissue (Taylor, G. et al.,1984, Infect. Immun. 43:649-655). Cotton rat models of RSV are also wellknown. Virus replicates to high titer in the nose and lungs of thecotton rat but produces few if any signs of inflammation. Additionalassays for evaluating the effectiveness of the modified viralpolypeptides are well known to those of ordinary skill in the art.

Pharmaceutical Compositions and Routes of Administration

As used herein, the compounds of this invention (e.g., the modifiedpolypeptides described herein), are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood) orwhich enhance delivery of the parent compound to a biologicalcompartment (e.g., the brain or lymphatic system) relative to the parentspecies. Preferred prodrugs include derivatives where a group whichenhances aqueous solubility or active transport through the gut membraneis appended to the structure of formulae described herein.

The compounds of this invention may be modified by appending appropriatefunctionalities to enhance selective biological properties. Suchmodifications are known in the art and include those which increasebiological penetration into a given biological compartment (e.g., blood,lymphatic system, central nervous system), increase oral availability,increase solubility to allow administration by injection, altermetabolism and alter rate of excretion. Pharmaceutically acceptablesalts of the compounds of this invention include those derived frompharmaceutically acceptable inorganic and organic acids and bases.Examples of suitable acid salts include acetate, adipate, benzoate,benzenesulfonate, butyrate, citrate, digluconate, dodecylsulfate,formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate,hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate,phosphate, picrate, pivalate, propionate, salicylate, succinate,sulfate, tartrate, tosylate and undecanoate. Salts derived fromappropriate bases include alkali metal (e.g., sodium), alkaline earthmetal (e.g., magnesium), ammonium and N-(alkyl)₄₊ salts. This inventionalso envisions the quaternization of any basic nitrogen-containinggroups of the compounds disclosed herein. Water or oil-soluble ordispersible products may be obtained by such quaternization.

The compounds of the invention can, for example, be administered byinjection, intravenously, intraarterially, subdermally,intraperitoneally, intramuscularly, or subcutaneously; or orally,buccally, nasally, transmucosally, intravaginally, cervically,topically, in an ophthalmic preparation, or by inhalation, with a dosageranging from about 0.001 to about 100 mg/kg of body weight, or accordingto the requirements of the particular drug and more preferably from0.5-10 mg/kg of body weight. The methods herein contemplateadministration of an effective amount of compound or compoundcomposition to achieve the desired or stated effect. Typically, thepharmaceutical compositions of this invention will be administered fromabout 1 to about 6 times per day or alternatively, as a continuousinfusion, or for example as an intravaginal foam or formulated for acervical ring if used singly or in combination with a contraceptive.Such administration can be used as a chronic or acute therapy. Theamount of active ingredient that may be combined with the carriermaterials to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. A typicalpreparation will contain from about 1% to about 95% active compound(w/w). Alternatively, such preparations contain from about 20% to about80% active compound.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular patient will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the patient'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a patient's condition or prevention of infection, amaintenance dose of a compound, composition or combination of thisinvention may be administered, if necessary. Subsequently, the dosage orfrequency of administration, or both, may be reduced, as a function ofthe symptoms, to a level at which the improved condition is retained.Patients may, however, require intermittent treatment on a long-termbasis upon any recurrence of disease symptoms (e.g. increase in HIVviral load).

Pharmaceutical compositions of this invention comprise a compounds ofthe invention or a pharmaceutically acceptable salt thereof; anadditional agent including for example, morphine or codeine; and anypharmaceutically acceptable carrier, adjuvant or vehicle. Alternatecompositions of this invention comprise a compound of the invention or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, adjuvant or vehicle. The compositions delineatedherein include the compounds of the invention delineated herein, as wellas additional therapeutic agents if present, in amounts effective forachieving a modulation of disease or disease symptoms, including HIVmediated disorders or symptoms thereof.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asd-α-tocopherol polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tween® or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as alpha-, beta-, and gamma-cyclodextrin,may also be advantageously used to enhance delivery of compounds of theformulae described herein.

The pharmaceutical compositions of this invention may be administeredenterally for example by oral administration, parenterally, byinhalation spray, topically, rectally, nasally, buccally, vaginally orvia an implanted reservoir, preferably by oral or vaginal administrationor administration by injection. The pharmaceutical compositions of thisinvention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases, or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, and intracranial injection orinfusion techniques.

Examples of dosage forms include, but are not limited to: tablets;caplets; capsules, such as soft elastic gelatin capsules; cachets;troches; lozenges; dispersions; suppositories; ointments; cataplasms(poultices); pastes; powders; dressings; creams; plasters; solutions;patches; aerosols (e.g., nasal sprays or inhalers); gels; liquid dosageforms suitable for oral or mucosal administration to a patient,including suspensions (e.g., aqueous or non-aqueous liquid suspensions,oil-in-water emulsions, or a water-in-oil liquid emulsions), solutions,and elixirs; liquid dosage forms suitable for parenteral administrationto a patient; and sterile solids (e.g., crystalline or amorphous solids)that can be reconstituted to provide liquid dosage forms suitable forparenteral administration to a patient.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween® 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant, or carboxymethyl cellulose or similar dispersing agentswhich are commonly used in the formulation of pharmaceuticallyacceptable dosage forms such as emulsions and or suspensions. Othercommonly used surfactants such as Tweens or Spans and/or other similaremulsifying agents or bioavailability enhancers which are commonly usedin the manufacture of pharmaceutically acceptable solid, liquid, orother dosage forms may also be used for the purposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, emulsions and aqueous suspensions,dispersions and solutions. In the case of tablets for oral use, carrierswhich are commonly used include lactose and corn starch. Lubricatingagents, such as magnesium stearate, are also typically added. For oraladministration in a capsule form, useful diluents include lactose anddried corn starch. When aqueous suspensions and/or emulsions areadministered orally, the active ingredient may be suspended or dissolvedin an oily phase is combined with emulsifying and/or suspending agents.If desired, certain sweetening and/or flavoring and/or coloring agentsmay be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

The pharmaceutical compositions of the invention may be administeredtopically or intravaginally. The pharmaceutical composition will beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. In still another embodiment, the pharmaceuticalcomposition is formulated as a vaginal ring. Suitable carriers include,but are not limited to, mineral oil, sorbitan monostearate, polysorbate60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcoholand water. The pharmaceutical compositions of this invention may also betopically applied to the lower intestinal tract by rectal suppositoryformulation or in a suitable enema formulation. Topically-transdermalpatches and iontophoretic administration are also included in thisinvention. In one embodiment, the compound of the invention isadministered vaginally as a prophylactic treatment for a sexuallytransmitted disease, e.g., HIV.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

When the compositions of this invention comprise a combination of acompound of the formulae described herein and one or more additionaltherapeutic or prophylactic agents, both the compound and the additionalagent should be present at dosage levels of between about 1 to 100%, andmore preferably between about 5 to 95% of the dosage normallyadministered in a monotherapy regimen. The additional agents may beadministered separately, as part of a multiple dose regimen, from thecompounds of this invention. Alternatively, those agents may be part ofa single dosage form, mixed together with the compounds of thisinvention in a single composition.

With respect to HIV, peptides of the invention may be used astherapeutics in the treatment of HIV infection and/or AIDS. In addition,the peptides may be used as prophylactic measures in previouslyuninfected individuals after acute exposure to an HIV virus (e.g.post-exposure prophylaxis). Examples of such prophylactic use of thepeptides may include, but are not limited to, prevention of virustransmission from mother to infant and other settings where thelikelihood of HIV transmission exists, such as, for example, sexualtransmission or accidents in health care settings wherein workers areexposed to HIV-containing blood products.

Effective dosages of the peptides of the invention to be administeredmay be determined through procedures well known to those in the artwhich address such parameters as biological half-life, bioavailability,and toxicity.

A therapeutically effective dose refers to that amount of the compoundsufficient to result in amelioration of symptoms or a prolongation ofsurvival in a patient. Toxicity and therapeutic efficacy of suchcompounds can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., for determining the LD₅₀(the dose lethal to 50% of the population) and the ED₅₀ (the dosetherapeutically effective in 50% of the population). The dose ratiobetween toxic and therapeutic effects is the therapeutic index and itcan be expressed as the ratio LD₅₀/ED₅₀. Compounds which exhibit largetherapeutic indices are preferred. The data obtained from these cellculture assays and animal studies can be used in formulating a range ofdosage for use in humans. The dosage of such compounds lies preferablywithin a range of circulating concentrations that include the ED50 withlittle or no toxicity. The dosage may vary within this range dependingupon the dosage form employed and the route of administration utilized.For any compound used in the method of the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC₅₀ (e.g., theconcentration of the test compound which achieves a half-maximalinhibition of the fusogenic event, such as a half-maximal inhibition ofviral infection relative to the amount of the event in the absence ofthe test compound) as determined in cell culture. Such information canbe used to more accurately determine useful doses in humans. Levels inplasma may be measured, for example, by high performance liquidchromatography (HPLC) or mass spectrometry (MS).

Prophylactic Vaccine

The peptides of the invention may, further, serve the role of aprophylactic vaccine, wherein the host raises antibodies against thepeptides of the invention, which then serve to neutralize a virus (e.g.,HIV, RSV, influenza, parainfluenza, coronavirus, ebolavirus) by, forexample, inhibiting further infection. Administration of the peptides ofthe invention as a prophylactic vaccine, therefore, would compriseadministering to a host a concentration of peptides effective in raisingan immune response which is sufficient to neutralize the virus, by, forexample, inhibiting virus ability to infect cells. The exactconcentration will depend upon the specific peptide to be administered,but may be determined by using standard techniques for assaying thedevelopment of an immune response which are well known to those ofordinary skill in the art. The peptides to be used as vaccines areusually administered intramuscularly.

The peptides may be formulated with a suitable adjuvant in order toenhance the immunological response. Such adjuvants may include, but arenot limited to mineral gels such as aluminum hydroxide; surface activesubstances such as lysolecithin, pluronic polyols, polyanions; otherpeptides; oil emulsions; and potentially useful human adjuvants such asBCG and Corynebacterium parvum. Many methods may be used to introducethe vaccine formulations described here. These methods include but arenot limited to oral, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, and intranasal routes.

Alternatively, an effective concentration of polyclonal or monoclonalantibodies raised against the peptides of the invention may beadministered to a host so that no uninfected cells become infected bythe virus. The exact concentration of such antibodies will varyaccording to each specific antibody preparation, but may be determinedusing standard techniques well known to those of ordinary skill in theart. Administration of the antibodies may be accomplished using avariety of techniques, including, but not limited to those described inthis section.

In one aspect, the invention is directed to a method of generating anantibody to a modified polypeptide. The method includes administering amodified polypeptide(s) of the invention to a subject so as to generatean antibody to the modified polypeptide.

In yet another aspect, the invention is directed to an antibody thatspecifically binds a modified polypeptide, wherein the modifiedpolypeptide has an amino acid sequence of any of the sequences of FIG.5, 6, or

the modified polypeptides include a heptad repeat domain with thesequence:

BTWXEWDXEINNYTSLIHSL (SEQ ID NO: 15),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 16),BTWBXWDRXINNYTSL (SEQ ID NO: 17),BTWBEWDREINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 18),BTWBXWDRXINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO: 19),BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE (SEQ ID NO: 20),BTWBEWDREINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 21),BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 22),BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 23),BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE (SEQ ID NO: 24),BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 25),BTWBXWDRXINNYTSXIHSXIEESQNQQXKNEXELLE (SEQ ID NO: 26),YTSXIHSXIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 27),YTSLIXSLIXESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 28),YTSLIHSLIEXSQNXQEKNEQELLELDKWASLWNWF (SEQ ID NO: 29),YTSLIHSLIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 30),YTSLIHSLIEESQNQQEXNEQXLLELDKWASLWNWF (SEQ ID NO: 31),YTSLIHSLIEESQNQQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 32),YTSLIHSLIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 33),YTSLIHSLIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 34),YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF (SEQ ID NO: 35),YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF (SEQ ID NO: 36),YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF (SEQ ID NO: 37),YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF (SEQ ID NO: 38),YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF (SEQ ID NO: 39),YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF (SEQ ID NO: 40),YTSXIHSXIEESQNQQXKNEXELLELDXWASXWNWF (SEQ ID NO: 41),BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE (SEQ ID NO: 42), orBTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE (SEQ ID NO: 43);wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine.Uses of the Modified Polypeptides

The antifusogenic capability of the modified peptides of the inventionmay additionally be utilized to inhibit or treat/ameliorate symptomscaused by processes involving membrane fusion events. Such events mayinclude, for example, virus transmission via cell-cell fusion andvirus-cell fusion. The peptides of the invention may be used to inhibitfree viral, such as retroviral, e.g., HIV, transmission to uninfectedcells wherein such viral infection involves membrane fusion events orinvolves fusion of a viral structure with a cell membrane.

In one aspect, the invention is directed to a method for inhibitingtransmission of HIV to a cell. The method includes contacting the HIVvirus with an effective dose of a modified polypeptide so that the HIVvirus is inhibited from infecting the cell. Preferably, the modifiedpolypeptide has a HIV gp41 heptad repeat domain (e.g., heptad repeatdomain 1 or 2, or combinations thereof) that is stabilized with ahydrocarbon staple. Suitable modified polypeptides include thosedirected to the heptad repeat domain 1, wherein the polypeptide is 30%or more identical to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:3or SEQ ID NO:14 and forms an alpha-helix. Other suitable modifiedpolypeptides include those directed to the heptad repeat domain 2,wherein the polypeptide is 30% or more identical to the amino acidsequence of FIG. 5, FIG. 6, SEQ ID NO:1 or 14 and forms an alpha-helix.

In yet another aspect, the invention is directed to a method fortreating or delaying the onset of AIDS in an HIV infected individual.The method entails administering to an individual infected with HIV aneffective dose of a pharmaceutical composition having a modifiedpolypeptide with a stabilized HIV gp41 heptad repeat domain, thustreating or delaying the onset of AIDS. Preferably the HIV gp41 heptadrepeat domain is stabilized with a hydrocarbon staple(s). Suitablepolypeptides include those directed to the heptad repeat domain 1,wherein the polypeptide is 30% or more identical to an amino acidsequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and forms analpha-helix. Other suitable polypeptides include those directed to theheptad repeat domain 2, wherein the polypeptide is 30% or more identicalto an amino acid sequence of FIG. 5, FIG. 6, SEQ ID NO:1 or 14 and formsan alpha-helix.

In still another aspect, the invention is directed to a method forincreasing the number of CD4+ cells in an individual infected with HIV.The method involves administering to the individual infected with HIV aneffective dose of a pharmaceutical composition having a modifiedpolypeptide with a stabilized HIV gp41 heptad repeat domain. Theadministration of the composition results in an increase in the numberof CD4+ cells in the individual. Preferably the HIV gp41 heptad repeatdomain is stabilized with a hydrocarbon staple(s). Suitable polypeptidesinclude those directed to the heptad repeat domain 1, wherein thepolypeptide is 30% or more identical to an amino acid sequence of SEQ IDNO:2, SEQ ID NO:3 or SEQ ID NO:14 and forms an alpha-helix. Othersuitable polypeptides include those directed to the heptad repeat domain2, wherein the polypeptide is 30% or more identical to an amino acidsequence selected of FIG. 5, FIG. 6, or SEQ ID NO:1 and forms analpha-helix.

In yet another aspect, the invention is directed to a method forinhibiting syncytia formation between an HIV infected cell and anuninfected cell. The method involves contacting the infected cell withan effective dose of a composition having a modified polypeptide with astabilized HIV gp41 heptad repeat domain, thereby inhibiting syncytiaformation between the cells. Preferably the HIV gp41 heptad repeatdomain is stabilized with a hydrocarbon staple. Suitable polypeptidesinclude those that are 30% or more identical to an amino acid sequenceof FIG. 5, FIG. 6, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:13,or SEQ ID NO:14 and forms an alpha-helix.

In still another aspect, the invention is directed to a method forinactivating HIV. The method involves contacting the virus with aneffective dose of a modified polypeptide having a stabilized HIV gp41heptad repeat domain so that the HIV is rendered inactive (e.g.,non-infectious). Preferably the HIV gp41 heptad repeat domain isstabilized with a hydrocarbon staple(s). Suitable polypeptides includethose that are 30% or more identical to an amino acid sequence of FIG.5, FIG. 6, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:1, SEQ ID NO:13 or SEQ IDNO:14 and forms an alpha-helix.

In still another aspect, the invention is directed to a method forpreventing an HIV infection in an individual. The method involvesadministering to an individual an effective dose of a pharmaceuticalcomposition having modified polypeptide with a stabilized HIV gp41heptad repeat domain, wherein the stabilized HIV gp41 heptad repeatdomain interferes with the ability of the HIV to infect the individual.Suitable polypeptides include those directed to the heptad repeat domain1, wherein the polypeptide is 30% or more identical to an amino acidsequence of SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:14 and forms analpha-helix. Other suitable polypeptides include those directed toheptad repeat domain 2, wherein the polypeptide is 30% or more identicalto an amino acid sequence of FIG. 5, FIG. 6, SEQ ID NO:1 or 13 and formsan alpha-helix.

In another aspect, the invention is directed to a method for inhibitingthe transmission of RSV to a cell. The method includes contacting thevirus with an effective dose of a modified polypeptide having astabilized RSV viral alpha helix heptad repeat-analog domain, therebyinhibiting transmission of the virus to a cell. Preferably the heptadrepeat domain is stabilized with a hydrocarbon staple(s) Suitablemodified polypeptides include those which are 30% or more identical toSEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:11 and SEQ ID NO:12 and forms analpha-helix.

In yet another aspect, the invention is directed to a method forinhibiting the transmission of influenza virus to a cell. The methodincludes contacting the virus with an effective dose of a modifiedpolypeptide having a stabilized influenza viral alpha helix heptadrepeat-analog domain, thereby inhibiting transmission of the virus to acell. Preferably the heptad repeat domain is stabilized with ahydrocarbon staple(s). Suitable polypeptides are known in the art.

In yet another aspect, the invention is directed to a method forinhibiting the transmission of a parainfluenza virus to a cell. Themethod includes contacting the virus with an effective dose of amodified polypeptide having a stabilized parainfluenza viral alpha helixheptad repeat-analog domain, thereby inhibiting transmission of thevirus to a cell. Preferably the heptad repeat domain is stabilized witha hydrocarbon staple(s). Suitable polypeptides include those which are30% or more identical to (SEQ ID NO:6) and forms an alpha-helix.

In still another aspect, the invention is directed to a method forinhibiting the transmission of a coronavirus to a cell. The methodincludes contacting the coronavirus with an effective dose of a modifiedpolypeptide having a stabilized coronavirus alpha helix heptadrepeat-analog domain, thereby inhibiting transmission of the virus to acell. Preferably the heptad repeat domain is stabilized with ahydrocarbon staple(s). Suitable polypeptides include those which are 30%or more identical to (SEQ ID NO:7) or (SEQ ID NO:8) and forms analpha-helix.

In yet still another aspect, the invention is directed to a method forinhibiting the transmission of an ebola virus to a cell. The methodincludes contacting the ebolavirus with an effective dose of a modifiedpolypeptide having a stabilized ebolavirus alpha helix heptadrepeat-analog domain, thereby inhibiting transmission of the virus to acell. Preferably the heptad repeat domain is stabilized with ahydrocarbon staple(s). Suitable polypeptides include those having anamino acid sequence which is 30% identical to (SEQ ID NO:9) or (SEQ IDNO:10) and forms an alpha-helix.

Preferably, any of the above modified polypeptides used in the methodsof the invention have the structure of Formula (I), (II) or (III) asdescribed herein.

Kits

The present invention also encompasses a finished packaged and labeledpharmaceutical product. This article of manufacture includes theappropriate unit dosage form in an appropriate vessel or container suchas a glass vial or other container that is hermetically sealed. Thepharmaceutical product may contain, for example, a compound of theinvention in a unit dosage form in a first container, and in a secondcontainer, sterile water for injection. Alternatively, the unit dosageform may be a solid suitable for oral, transdermal, intranasal,intravaginal, cervical ring, or topical delivery.

In a specific embodiment, the unit dosage form is suitable forintravenous, intramuscular, intranasal, oral, intravaginal, cervical,topical or subcutaneous delivery. Thus, the invention encompassessolutions, solids, foams, gels, preferably sterile, suitable for eachdelivery route.

As with any pharmaceutical product, the packaging material and containerare designed to protect the stability of the product during storage andshipment. Further, the products of the invention include instructionsfor use or other informational material that advise the physician,technician, or patient on how to appropriately prevent or treat thedisease or disorder in question. In other words, the article ofmanufacture includes instruction means indicating or suggesting a dosingregimen including, but not limited to, actual doses, monitoringprocedures (e.g. detection and quantitation of infection), and othermonitoring information.

Specifically, the invention provides an article of manufacturecomprising packaging material, such as a box, bottle, tube, vial,container, sprayer, insufflator, intravenous (i.v.) bag, envelope andthe like; and at least one unit dosage form of a pharmaceutical agentcontained within said packaging material, wherein said pharmaceuticalagent comprises a compound of the invention, and wherein said packagingmaterial includes instruction means which indicate that said compoundcan be used to prevent, manage, treat, and/or ameliorate one or moresymptoms associated with a viral disease by administering specific dosesand using specific dosing regimens as described herein.

The following examples are provided merely as illustrative of variousaspects of the invention and shall not be construed to limit theinvention in any way.

EXAMPLES Example 1 Synthesis of Hydrocarbon Stapled Alpha HelicalPolypeptides

A combined strategy of structural analysis and chemical synthesis isapplied to construct the modified polypeptides. Asymmetric syntheses ofα,α-disubstituted amino acids is first performed as previously reported(Schafmeister, C. E., J. Po, and G. L. Verdine, Journal of the AmericanChemical Society, 2000. 122(24): p. 5891-5892; Walensky, L. D., et al.,Science, 2004. 305(5689): p. 1466-1470). The modified polypeptidecompounds are generated by replacing at least two naturally occurringamino acids with the α,α-disubstituted non-natural amino acids atdiscrete locations flanking either 2, 3 or 6 amino acids, namely the “i,i+3,” “i, i+4” or “i, i+7” positions, respectively.

Locations for the non-natural amino acids and subsequent hydrocarbonstaple(s) are carefully chosen so as not to interfere with N36interactions (Chan, D. C., et al., Cell, 1997. 89(2): p. 263-273).Residues in positions a and d interact directly with N36, whereas,residues e and g may contact the N36 core as a result of the pitch ofthe six-helix bundle. Residues b, f, and c localize to the opposite faceof the α-helix and are thus ideally located for placement of thehydrocarbon staple(s).

The modified polypeptides can be generated using solid phase Fmocchemistry and ruthenium-catalyzed olefin metathesis, followed by peptidedeprotection and cleavage, purification by reverse-phase highperformance liquid chromatography, and chemical characterization usingLC/MS mass spectrometry and amino acid analysis.

Alternatively an established fragment-based approach can be pursued([Bray, B. L. Nature Reviews Drug Discovery, 2003. 2(7): p. 587-593;MYUNG-CHOL KANG, B. B., et al., Methods and compositions for peptidesynthesis, U.S.P.a.T. Office, Editor. Jan. 18, 2000 USA). In thisstrategy, the peptide is divided into 3 fragments, such that anN-terminal, central, and C-terminal portion are synthesizedindependently. These polypeptide fragments should be generated usingsolid phase Fmoc chemistry and ruthenium-catalyzed olefin metathesis onsuper-acid cleavable resins, which will yield fully protected peptideshaving an Fmoc at the N-terminus, and either a C-terminal amide (for theC-terminal fragment) or a free carboxylate (for the central andN-terminal fragments). These fully protected fragments are purified byreverse-phase high performance liquid chromatography, followed bysequential deprotection, coupling, and purification, to yield the fulllength, fully protected polypeptides. Global deprotection, followed byreverse-phase high performance liquid chromatography will yield thefinal products, which can be characterized using LC/MS mass spectrometryand amino acid analysis.

Example 2 Determining the Secondary Structure and Proteolytic Stabilityof the Modified Polypeptides

The α-helicity of stapled modified polypeptides can be compared to theirunmodified counterparts by circular dichroism. CD spectra can beobtained on a Jasco J-710 or Aviv spectropolarimeter at 20° C. using thefollowing standard measurement parameters: wavelength, 190-260 nm; stepresolution, 0.5 nm; speed, 20 nm/sec; accumulations, 10; response, 1sec; bandwidth, 1 nm; path length, 0.1 cm. The α-helical content of eachpeptide is calculated by dividing the mean residue ellipticity[Θ]222_(obs) by the reported [Θ]222_(obs) for a model helical peptide(Forood, B., E. J. Feliciano, and K. P. Nambiar, PNAS, 1993. 90(3): p.838-842; J. Martin Scholtz, Biopolymers, 1991. 31(13): p. 1463-1470;Lawless, M. K., et al., Biochemistry, 1996. 35(42): p. 13697-13708) orusing, for example, the Aviv machine using CDNN software developed byBrohm in order to deduce five different secondary structure fractions(helix, parallel and antiparallel beta-sheet, beta-turn and randomcoil). Protein Engineering, 1992. 5(3); p. 191-195

To assess whether helix stabilization confers enhanced proteaseresistance and serum stability, the modified polypeptides can besubjected to trypsin/chymotrypsin degradation assays and in vitro and invivo serum stability assays, and compared to their unmodifiedcounterparts as previously described (Walensky, L. D., et al., Science,2004. 305(5689): p. 1466-1470). Recovery of intact compound isdetermined, for example, by flash freezing the in vitro or serumspecimens in liquid nitrogen, lyophilization, and extraction in 50:50acetonitrile/water containing 0.1% trifluoroacetic acid, followed byLC/MS based detection and quantitation.

Example 3 Optimization of the Biophysical and Biochemical Properties ofthe Modified Polypeptides by Evaluating Diversified Modified PeptideLibraries Synthesized in High-Throughput Fashion

High-throughput technologies can be used to optimize the modifiedpolypeptides activities for cellular and in vivo studies. For example,an Apex 396 multichannel synthesizer (AAPPTEC; Louisville, Ky.) can beused to produce polypeptide libraries for biological evaluation. Thepolypeptide compounds can be diversified by extension, truncation, oramino acid substitution across natural and select non-natural aminoacids, and differential staple localization can be made to maximizetheir biophysical and biochemical properties. The libraries aregenerated using high-throughput solid phase Fmoc chemistry andruthenium-catalyzed olefin metathesis and peptide deprotection andcleavage. Peptide purification is achieved by reverse phase C18 HPLC,and products characterized by LC/MS mass spectrometry and amino acidanalysis.

Example 4 Evaluating the Modified Polypeptides Ability to Target andInhibit HIV Fusion

The binding activity and functional effects of the HIV modifiedpolypeptides can be assessed in fluorescence polarization, syncytialfusion, and HIV infectivity assays. Equilibrium binding constants can bedetermined by fluorescence polarization assays (FPA) using fluoresceinisothiocyanate (FITC)-labeled modified polypeptides and titratedrecombinant five-helix bundle protein. FPA experiments can be performedusing a BMG Labtech FLUOstar optima microplate reader, and dissociationconstants determined by regression analysis using GraphPad software(Prism). The recombinant 5-helix bundle protein, first developed by Rootet al., contains five of the six helices that comprise the core of thegp41 trimer-of-hairpins, which are connected by short peptide linkers(Root, M. J., M. S. Kay, and P. S. Kim, Science, 2001. 291(5505): p.884-888). Because the 5-helix bundle lacks the third C-peptide helix andunder experimental conditions is soluble, stable, and helical,incorporation of the sixth C-peptide in the form of FITC-modifiedpolypeptide would provide a direct measure of binding activity. In thismanner, modified polypeptides, differing in peptide sequence, staplelocation, and staple number, can be screened for maximal in vitrobinding activity. Binding activity can also be determined indirectly bycompetition assays in which the 5-helix bundle is combined with aFITC-labeled unmodified HIV fusion inhibitor peptide and then unlabeledstapled gp41 peptides are added at increasing concentrations followed bymeasurement of fluorescence polarization and then calculation of Ki bynonlinear regression analysis, as indicated above.

Alternatively, an alternative binding assay can be employed based uponthe “gp41-5” construct of Frey et al. Gp41-5 binds with high affinity toadded peptides that contain all or part of the missing CHR. For example,using gp41-5 and fluorescein-labeled C38 (residues 117-154), Frey et al.successfully generated an FPA binding curve that revealed a K_(d) of 3.6nM (Frey, G., et al., PNAS, 2006. 103(38): p. 13938-13943).

Functional assays can also be used to evaluate the modified polypeptidesactivity. In culture, multinucleated giant cells or “syncytia” form as aresult of direct cell-cell fusion between HIV-1-infected and uninfectedCD4-positive cells. In the syncytia formation assay, an indicator cellline expressing the CD4 receptor, and a fusogenic cell line that lacksthe CD4 receptor but contains HIV-1 proteins on the surface, fuse togenerate 70-100 multinucleated giant cells in culture within 48 h.Syncytia are then counted using an inverted microscope. The ability ofstabilized alpha helix of gp41 (SAH-gp41) compounds to inhibit syncytiaformation in a dose-responsive fashion is used as a functional measureof fusion inhibition, for which IC₅₀s can be determined and comparedwith peptides T20 and T649 (Brenner, T. J., et al. The Lancet, 1991.337(8748): p. 1001-1005; Madani, N., et al., Journal of Virology, 2007.81(2): p. 532-538).

Also the anti-viral properties of the modified polypeptides can bequantified based upon their capacity to directly block HIV infection ofCD4-positive and CCR5-expressing canine thymus cells. Recombinant HIV-1viruses (eg. HXBc2, YU2, and additional strains available through theNIH AIDS Research and Reference Reagent Program) expressing fireflyluciferase and containing the indicated envelope glycoproteins can beused to infect Cf2Th-CD4-CCR5/CXCR4 cells in the presence of seriallydiluted HIV modified polypeptides. After 48 hours, the cells are lysedand luciferase activity is quantified (Si, Z. H., M. Cayabyab, and J.Sodroski, Journal of Virology, 2001. 75(9): p. 4208-4218 Si, Z. H., etal., PNAS, 2004. 101(14): p. 5036-5041). The identical experiment isperformed with the amphotropic murine leukemia virus (AMLV), to monitorfor any nonspecific effects of the modified polypeptides. Similarcontrol assays may be performed with non-HIV modified polypeptides ofthe invention and are known in the art.

Example 5 Evaluate the Ability of SAH-gp41 Compounds to OvercomeResistance to Enfuvirtide

Heavily antiretroviral-treated HIV-positive patients who respond toinitial treatment with enfuvirtide, may ultimately develop a viralrebound that typically occurs within a maximum of 80 weeks. Resistanceto enfuvirtide derives from mutations within the HR-1 region of gp41,although some genetic changes are found in the HR-2 domain (Xu, L., etal., Antimicrobial Agents and Chemotherapy, 2005. 49(3): p. 1113-1119;Perez-Alvarez, L., et al. Journal of Medical Virology, 2006. 78(2): p.141-147). These mutations, such as I37V, V38A/E/M, Q39R, Q40H, N42T/Q/H,N43D/Q, are only found in enfuvirtide-experienced patients (Poveda, E.,et al., Journal of Medical Virology, 2004. 74(1): p. 21-28; Melby, T.,et al., Aids Research and Human Retroviruses, 2006. 22(5): p. 375-385;Sista, P. R., et al., Aids, 2004. 18(13): p. 1787-1794; Wei, X. P., etal., Antimicrobial Agents and Chemotherapy, 2002. 46(6): p. 1896-1905).

Structural analysis and molecular modeling can be used to evaluate theimpact of these mutations on the binding interface of the HR-1 domainwith enfuvirtide. Five-helix bundle proteins incorporating resistancemutations can then be generated for binding analysis as described inExample 4. FITC-labeled SAH-gp41 compounds can then be screened againstthese mutant five-helix bundle proteins to determine if any nativeSAH-gp41 compounds retain activity despite HR domain mutations.Alternatively, HR1 peptides that contain resistance mutations aresynthesized and can be directly incubated with SAH-gp41 compounds, andthen run on native gels to detect and quantitate the formation ofheteroduplexes, which represent HR1-SAH-gp41 complex, detectable byfluorescence scanning of the gel (FIG. 20A). SAH-gp41 compounds shouldcontain T649 sequences known to contact two gp41 residues (Leu-568 andTrp-571) that are critical for fusion activity. By incorporating thissequence functionality, the SAH-gp41 compounds may overcomeenfuvirtide-resistant virus and are less likely to elicit a resistantvirus, in contrast to analogs, like T20, that lack such residues at theN-terminal region of the HR-2 domain (Cao, J., et al., Journal ofVirology, 1993. 67(5): p. 2747-2755; Chan, D. C., C. T. PNAS 1998.95(26): p. 15613-15617; Rimsky, L. T., D. C. Shugars, and T. J.Matthews, J. Virol., 1998. 72(2): p. 986-993). Follow-up HIV infectivitystudies would evaluate the functional activity of such SAH-gp41compounds against the corresponding primary resistant isolates.

To monitor for restoration of SAH-gp41 activity, FITC labeled mutantsSAH-gp41 (mSAH-gp41) compounds can be screened for binding affinity tomutant five-helix bundle proteins and for suppression of HIV infectivityusing primary resistance strains.

To explore the evolution of potential resistance to SAH-gp41 compounds,HIV strains can be evolved in the presence of increasing concentrationsof lead SAH-gp41 compounds. Resistant strains can be genotyped forcomparative mutational analysis between these mutants andenfuvirtide-resistant mutants (Van Laethem, K., et al., Journal ofVirological Methods, 2005. 123(1): p. 25-34). Because resistance to onetype of entry inhibitor may not affect susceptibility to other variants,(Ray, N., et al., Journal of Virology, 2007. 81(7): p. 3240-3250)combined SAH-gp41 and mSAH-gp41 polypeptide compositions can beformulated.

Alternative, a phage display strategy can be employed. Lai et al.successfully used phage display to restore heterodimerization of acoiled-coil pair of α-helices after destabilizing mutations wereintroduced (Lai, J. R., et al., Journal of the American ChemicalSociety, 2004. 126(34); p. 10514-10515). Whereas complimentaryelectrostatic pairing preferences among helical residues that flank thecore are readily apparent, less is known about the packing preferencesof non-polar residues located at core positions (Lumb, K. J. and P. S.Kim, Science, 1995. 268(5209): p. 436-439) Using phage display, one canscreen all possible amino acid combinations at up to 7 variablelocations of the HR-2 domain for binding affinity to a mutant HR-1domain, using the corresponding five-helix bundle. In addition, phagedisplay screening of fully randomized HR-2 domains against combinationsof known mutations in HR-1 domains could be undertaken in order todetermine the SAH-gp41 sequence capable of forming the most stablecomplex with the 5-helix bundle (Xu, L., et al., Antimicrobial Agentsand Chemotherapy, 2005. 49(3): p. 1113-1119; Perez-Alvarez, L., et al.,Journal of Medical Virology, 2006. 78(2): p. 141-147). After threecycles of “panning”, phage DNA sequencing would reveal those peptidesequences having the highest binding affinities for the mutant 5-helixbundle. The corresponding SAH-gp41 derivatives would then be synthesizedand evaluated in binding and activity studies as described above.

Example 6 Analyze the In Vivo Stability, Pharmacokinetics, andBiodistribution of SAH-gp41 Compounds

A rigorous assessment of the in vivo pharmacology of SAH-gp41 compoundscan be used to determine and optimize the therapeutic potential of themodified polypeptides. For in vivo serum half-life studies, 5-50 mg/kgof FITC-labeled or unlabeled SAH-gp41 polypeptides can be injected ordelivered orally into control mice and blood specimens withdrawn forexample at 0, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 hours post-injection tomeasure levels of intact compound by HPLC as previously described(Walensky, L. D., et al., Science, 2004. 305(5689): p. 1466-1470]) or byreverse-phase LC/MS, followed by mathematical determination ofpharmacokinetic parameters using formulas and software known in the art.LC/MS-based characterization of metabolites can also be performed.111In-DOTA-derivatized compounds can be synthesized and injected intocontrol mice for measurement of tissue uptake, excretion, andbiodistribution of the modified polypeptide compounds over time byradioisotope scintillation counting. SPECT/NMR imaging of control miceinjected with 111In-DOTA-derivatized modified compounds will providehigh resolution images of biodistribution in real time as previouslyperformed by the Walensky lab in collaboration with Ralph Weissleder,M.D. of Massachusetts General Hospital (Hird V, V. M., et al. Br JCancer, 1991. 64(5): p. 911-4). Chemical modifications, includinglipidation, polysialylation, or antibody-conjugation, could be performedshould optimization of pharmacokinetics and tissue targeting of modifiedcompounds.

Example 7 Native gp41 C-Terminal Heptad Peptides are PredominantlyRandom Coils in Solution

gp41 HR-2-derived peptides based upon the sequences of T20 (residues638-673) and a T649 variant, T649v (residues 626-662, rather than T649is 628-663) were prepared and the circular dichroism (CD) spectradetermined at physiologic pH. The native peptides display only modestminima at 222 nm and 208 nm, suggesting predominantly random coilstructure in solution. Indeed, the calculated α-helical content (Forood,B., E. J. Feliciano, and K. P. Nambiar, PNAS, 1993. 90(3): p. 838-84; J.Martin Scholtz, Biopolymers, 1991. 31(13): p. 1463-1470; Lawless, M. K.,et al., Biochemistry, 1996. 35(42): p. 13697-13708) was only ˜25% forT20 and 14% for T649v. Thus, synthetic gp41-derived HR-2 peptides arepredominantly disordered in solution, reflecting a significant loss ofbioactive structure.

Example 8 Truncated C-Terminal Heptad Peptides Display Enhancedα-Helicity Upon Incorporation of an All-Hydrocarbon Staple

In order to improve the biochemical properties of HIV gp41-HR-2 peptidesthe T649v peptide was truncated to yield a 20-mer consisting of residues626-645 (FIG. 7). The truncated SAH-gp41 compound, SAH-gp41(626-645)(A),was successfully synthesized in high yield. Analysis of comparative CDspectra revealed marked enhancement of α-helical content forSAH-gp41(626-645)(A) compared to its unstapled counterpart (48% vs.20%). Evaluation of the compounds in an HIV syncytial formation assayrevealed markedly enhanced inhibitory activity of SAH-gp41(626-645)(A)compared to its unstapled derivative. Thus, in spite of eliminating morethan 40% of the residues of T649v, the hydrocarbon staple successfullytransformed a 20-mer gp41 truncation with little α-helicity and onlymodest anti-syncytial activity, into an α-helical compound withsignificant structural stabilization and potent anti-syncytial activity(IC90, ˜100 nM) (FIG. 18).

The activity of SAH-gp41(626-645)(A) peptide was compared to theclinically approved T20 peptide in an HIV infectivity assay using theHXBc2 strain. The SAH-gp41(626-645)(A) displayed significant anti-HXBc2activity, particularly given the markedly shortened construct.

Example 9 SAH-gp41 compounds demonstrate marked α-helical stabilization,proteolytic stability, thermal stability, 5-helix bundle bindingaffinity

To optimize the activity of tSAH-gp41 peptides, an alternative strategybased upon inserting one or more hydrocarbon staples into thefull-length gp41-HR-2 constructs was pursued (FIG. 11, 12). Unmodifiedenfuvirtide and T649v were predominantly unstructured in pH 7 aqueoussolution at 21° C., exhibiting less than 20% α-helicity (FIG. 14A,B).All stapled derivatives displayed comparatively increased α-helicalcontent, with up to 4.7-fold structural stabilization (FIG. 14A-C). Theinsertion of either one or two hydrocarbon staples consistentlytransformed the circular dichroism spectra from a random coil patternwith a predominant single minimum at 204 nm to an α-helical contour withdouble minima at 208 and 222 nm. For select peptide templates, singleC-terminal stapling conferred a greater degree of α-helicalstabilization than single N-terminal stapling. Select doubly stapledSAH-gp41 compounds exhibited an intermediate enhancement in α-helicalstructure, balancing the effects of the N- and C-terminal singly stapledpeptides. Enhancement of peptide α-helicity was likewise observed atpH2, and in most cases, SAH-gp41 compounds were even more helical at pH2than at pH7 (FIG. 14D-F).

To assess the resistance of SAH-gp41 peptides to thermal unfolding, weperformed circular dichroism studies across a 1-91° C. temperaturerange. We observed that select single and double stapling of HIV-1fusion inhibitor peptides conferred α-helical stabilization that wasremarkably heat-resistant, sustaining an up to 2.3-fold enhancement inα-helicity even at 91° C. (FIG. 15).

A major limitation of peptides as therapeutics is their susceptibilityto rapid proteolytic degradation. Biologically active peptides such asenfuvirtide that are lengthy, unfolded, and replete with protease sitesare particularly vulnerable. One of the potential benefits of a covalentcrosslinking strategy to enforce peptide α-helicity is shielding of thevulnerable amide bonds from proteolysis. Because proteases require thatpeptides adopt an extended conformation to hydrolyze amide bonds, thestructural constraint afforded by the hydrocarbon staple can rendercrosslinked peptides protease-resistant. To determine if hydrocarbonstapling, and especially double stapling, could protect the 36 to 37-merHIV-1 fusion peptides from proteolysis, we subjected enfuvirtide, T649v,and SAH-gp41 peptides to direct protease exposure in vitro. Toespecially challenge the stapled peptides, we selected chymotrypsin,which can cleave gp41 HR2 peptides at numerous consensus cleavage sites,including 9-11 locations for SAH-gp41(638-673) and 7 locations forSAH-gp41(626-662).

In the presence of 0.5 ng/μL chymotrypsin, enfuvirtide and T649v (25 μM)exhibited rapid degradation, with half-lives of 12 and 14 minutes,respectively (FIG. 16A-C). In comparison, singly stapled SAH-gp41compounds displayed longer half-lives that ranged from 21 to 200minutes. The majority of doubly stapled compounds markedly surpassedtheir singly stapled counterparts, with select doubly stapled peptidesachieving half-lives of up to 1275 minutes. In most cases, doublestapling had a stronger influence on proteolytic stability than overallpeptide α-helicity, as select doubly stapled peptides had lowerα-helicity than select singly stapled peptides, but still exhibitedsuperior protease resistance. Almost all stapled peptides had theidentical number of chymotrypsin cleavage sites as the correspondingunmodified peptides, emphasizing that the observed protease resistancederived from peptide stapling itself, rather than elimination ofcleavage sites.

Peptides have poor oral bioavailability in part due to rapid acidhydrolysis in the proximal digestive tract. The compelling proteaseresistance of doubly stapled SAH-gp41 compounds at neutral pH promptedus to explore their stability under acidic conditions. In each case,acidification of the peptide solutions significantly enhanced theirα-helical content as measured by CD (FIG. 16D-F). Upon exposure topepsin at 0.5 ng/μL, enfuvirtide and T649v (25 μM) exhibited rapiddegradation, with half-lives of 4 and 11 minutes, respectively. Selectdoubly stapled SAH-gp41 compounds displayed half-lives ranging fromapproximately 80-800-fold greater than the unmodified peptides, andconsistently surpassed their singly stapled counterparts. Remarkably,select doubly-stapled SAH-gp41 peptides remained 80% intact afterexposure to pepsin at pH 2 for more than 12 hours. As observed forchymotrypsin resistance, double stapling itself, rather than overallpeptide α-helicity or number of cleavage sites, correlated with thesuperior resistance to pepsin hydrolysis. These studies highlight thecapacity of double stapling to generate HIV-1 fusion inhibitor peptideswith unprecedented resistance to proteolytic hydrolysis at both neutraland acidic pH.

The compounds of the invention were also measured for their affinity togp41 in a five-helix binding assay as described herein. As shown in FIG.17 the modified compounds bound substantially better than the unmodifiedcontrol polypeptides.

Example 10 SAH-gp41 Compounds Demonstrate Anti-Syncytial FormationActivity and Anti-HIV Viral Fusion Activity

The compounds of the invention were assayed for inhibition of syncytialformation using methods well known to those skilled in the art. Theresults of the assay are shown in FIG. 18. Equal amounts of eitherT20/gp41₍₆₃₈₋₆₇₃₎ or SAH-gp41₍₆₂₆₋₆₄₅₎A were added to the media. Asshown, the modified compounds inhibited syncytial formation more so thanunmodified control polypeptides.

In order to determine the functional impact of hydrocarbon-stapling ongp41-based fusion inhibitor activity, SAH-gp41 compounds were tested andcompared to their unmodified counterparts in a luciferase-based HIVinfectivity assay (Si, Z. H., M. Cayabyab, and J. Sodroski, Journal ofVirology, 2001. 75(9): p. 4208-4218; Si, Z. H., et al., PNAS, 2004.101(14): p. 5036-5041). Recombinant HIV-1 bearing the envelopeglycoproteins from three distinct HIV-1 strains, HXBc2, ADA, and HXBc2P3.2, and a negative control virus bearing the amphotropic murineleukemia virus (A-MLV) envelope glycoproteins, were evaluated. Comparedto enfuvirtide, select SAH-gp41(638-673) peptides exhibited a 3- to15-fold enhancement of inhibitory activity across all three HIV-1strains (FIG. 19). T649v, an HR2 peptide that encompasses a 37-aminoacid fragment terminating 11 residues upstream of enfuvirtide'sC-terminus, displayed 26-, 40-, and 16-fold greater inhibitory activitythan enfuvirtide against viruses with the HXBc2, ADA, and HXBc2P 3.2envelope glycoproteins, respectively. Given the marked potency of T649vagainst these viral strains, we found that the corresponding SAH-gp41peptides showed essentially comparable activity in infectivity assays.In order to probe for differential anti-viral potencies amongT649v-based stapled peptides, we screened the compounds against viruseswith envelope glycoproteins derived from the more resistant primary R5isolate, YU2. Compared to T649v, select SAH-gp41(626-662) peptidesdemonstrated enhanced anti-YU2 activity (FIG. 19, 20B). The ability ofSAH-gp41 peptides to overcome HIV-1 HR1 resistance mutations, wasfurther underscored by the superior binding activity of select SAH-gp41peptides to mutant HR1 peptides, as compared to unmodified gp41-basedfusion peptides, when assayed by fluorescence scan of electopheresedmixtures of HR1 and HR2/SAH-gp41 peptides (FIG. 20A).

These functional data reveal that insertion of one or more hydrocarbonstaples can yield SAH-gp41 peptides with potent and broad anti-HIV-1activity. The importance of striking a balance between α-helicalstabilization, proteolytic stability, and anti-viral activity isunderscored by the doubly stapled SAH-gp41(626-662)(A, F) peptide, whichcombines intermediate α-helical stabilization, the strikinganti-proteolysis feature of double stapling, and potent anti-viralactivity, to yield a pharmacologically optimized HIV-1 fusion inhibitorpeptide.

Example 11 A Doubly Stapled SAH-gp41 Peptide Demonstrates StrikingEnhancement of In Vivo Stability and Bioavailabilty Compared to theCorresponding Unmodified Peptide

Male C57/BL6 mice were administered intravenously or by oral gavage 10mg/kg of either SAH-gp41₍₆₂₆₋₆₆₂₎(A,F) or the corresponding unmodifiedpeptide. Blood samples withdrawn at 30 minutes by retro-orbital bleedwere subjected to quantitation using LC/MS-based blood tests. The levelof SAH-gp41₍₆₂₆₋₆₆₂₎(A,F) measured in the blood was more than 6-foldgreater than the measured level of the corresponding unmodified peptide.Strikingly, 30 minutes after oral administration, intactSAH-gp41₍₆₂₆₋₆₆₂₎(A,F) was detected in the blood at measurable levels,whereas the unmodified peptide was undetectable (FIG. 21). These dataemphasize that hydrocarbon stapling confers unique pharmacologicproperties to gp41-based fusion peptide sequences, enhancing their invivo stability and even conferring measurable oral bioavailability. Thissingle dose experiment demonstrates that the SAH-gp41 peptides could bedosed at a level to provide serum levels of the compound comparable tothe level of an unmodified peptide (e.g., enfuvirtide) suggesting that atherapeutically effective dose could be administered orally.

All patents, patent applications, GenBank numbers, and publishedreferences cited herein are hereby incorporated by reference in theirentirety as if they were incorporated individually. While this inventionhas been particularly shown and described with references to preferredembodiments thereof, it will be understood by those skilled in the artthat various changes in form and details may be made therein withoutdeparting from the scope of the invention encompassed by the appendedclaims.

What is claimed is:
 1. A modified polypeptide for oral administration toa subject comprising a stabilized alpha helix of HIV gp41 heptad repeatdomain, wherein said stabilized HIV gp41 heptad repeat domain isstabilized with two hydrocarbon staples, wherein said hydrocarbonstaples are positioned so as to link amino acid residues i and i+4, andwherein said polypeptide is detectable intact in the blood of a subject30 minutes after oral administration of said polypeptide to saidsubject.
 2. The modified polypeptide of claim 1, wherein said modifiedpolypeptide is 20 or more amino acids.
 3. The modified polypeptide ofclaim 1, wherein said heptad repeat domain comprises the formula:--W--W---I--Y---I--L---S--Q---N--E---L, or conservative amino acidsubstitutions thereof and wherein “-” can be any amino acid (SEQ ID NO:44).
 4. The modified polypeptide of claim 1, wherein said heptad repeatdomain comprises the formula: -TW--WDR-I--Y---I--LI---Q--QEK-E--L-EL, orconservative amino acid substitutions thereof and wherein “-” can be anyamino acid (SEQ ID NO: 45).
 5. The modified polypeptide of claim 1,wherein said modified polypeptide has at least 10%, 20%, 30%, 50%, 60%,70%, 80%, or 90% alpha helicity in aqueous solution as determined bycircular dichroism.
 6. The modified polypeptide of claim 1, wherein saidheptad repeat domain is an HIV-1 gp41 heptad repeat domain 1, an HIV-1gp41 heptad repeat domain 2, an HIV-2 gp41 heptad repeat domain 1, or anHIV-2 gp41 heptad repeat domain
 2. 7. The modified polypeptide of claim1, wherein said modified polypeptide is a chimera.
 8. The modifiedpolypeptide of claim 7, wherein said chimera has the amino acid sequenceof WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ ID NO: 46).
 9. Themodified polypeptide of claim 1, wherein said heptad repeat domain formsan alpha helix and is 30% or more identical to the amino acid sequenceselected from the group consisting of:YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:1),NNLLRAIEAQQHLLQLTVWGIKQLQARILAVERYLQDQ (SEQ ID NO:2),BTWBEWDREINNYTSLIHSL (SEQ ID NO:3),MTWMEWDREINNYTSLIHSLIEESQNQQEKNEQELLE (SEQ ID NO:13),YTHIIYSLIEQSQNQQEKNEQELLALDKWASLWNWF (SEQ ID NO:58), andMTMKWEREIDNYTHIIYSLIEQSQNQQEKNEQELLA (SEQ ID NO:59).
 10. The modifiedpolypeptide of claim 1, wherein said modified polypeptide has a formulaselected from the group consisting of: (SEQ ID NO: 20)BTWBXWDRXINNYTSLIHSLIEXSQNXQEKNEQELLE, (SEQ ID NO: 22)BTWBXWDRXINNYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 23)BTWBEWDXEINXYTSLIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 24)BTWBEWDREINXYTSXIHSLIEESQNQQXKNEXELLE, (SEQ ID NO: 25)BTWBEWDREINNYTSXIHSXIEESQNQQXKNEXELLE, (SEQ ID NO: 35)YTSLIHSLIEXSQNXQEKNEQXLLEXDKWASLWNWF, (SEQ ID NO: 36)YTSXIHSXIEESQNQQEKNEQELLELDKWXSLWXWF, (SEQ ID NO: 37)YTSLIHSLIEESQNQQXKNEXELLELDKWXSLWXWF, (SEQ ID NO: 38)YTSXIHSXIEESQNQQXKNEXELLELDKWASLWNWF, (SEQ ID NO: 39)YTSXIHSXIEESQNQQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 40)YTSLIHSLIEXSQNXQEKNEQELLELDXWASXWNWF, (SEQ ID NO: 42)BTWBXWDRXINNYTSLIHSLIEESQNQXEKNXQELLE, (SEQ ID NO: 43)BTWBXWDRXINNYTSLIHSLIEESQNXQEKXEQELLE;  and (SEQ ID NO: 82)BTWXEWDXEINNYTSLIHSLIEESQNQXEKNXQELLE;

wherein X is any amino acid and further identifies the amino acidresidues which are linked by a hydrocarbon staple, and B is methionineor norleucine.
 11. A kit comprising the modified polypeptide of claim 1and instructions for use.